Compositions for alleviating inflammation and oxidative stress in a mammal

ABSTRACT

An antioxidant-promoting composition that increases antioxidant defense potential in a subject is disclosed comprising  Bacopa monniera  extract; milk thistle extract, ashwagandha powder, green tea extract,  Gotu kola  powder,  Ginko biloba  leaf extract;  Aloe vera  powder; turmeric extract; and N-acetyl cysteine. The antioxidant-promoting composition of the invention safely induces cellular antioxidant potential to achieve an overall net decrease in oxidative stress without the undesirable side-effects associated with the individual components of the antioxidant-promoting composition. Also disclosed is a method for reducing the undesirable side-effects of free radicals in a subject by administering to a subject in need of such antioxidants an effective amount of antioxidant-promoting composition of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/216,514, filed Aug. 31, 2005 now U.S. Pat. No. 7,579,026, which is acontinuation of U.S. application Ser. No. 11/088,323, filed Mar. 23,2005, now U.S. Pat. No. 7,241,461, U.S. Pat. No. 7,241,461 claims thebenefit of priority under 35 U.S.C. §120 from U.S. Application Ser. No.60/555,802, filed on Mar. 23, 2004; U.S. Application Ser. No.60/590,528, filed on Jul. 23, 2004; U.S. Application Ser. No.60/604,638, filed on Aug. 26, 2004; U.S. Application Ser. No.60/607,648, filed on Sep. 7, 2004; U.S. Application Ser. No. 60/610,749,filed on Sep. 17, 2004; U.S. Application Ser. No. 60/643,754, filed onJan. 13, 2005, and U.S. Application Ser. No. 60/646,707, filed on Jan.25, 2005, the contents of which are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to compositions for alleviatinginflammation and oxidative stress in a subject. Specifically, thepresent invention is directed to the field of natural remedies and thedevelopment of compositions to increase the antioxidant potential of asubject by inducing the subject's natural cellular defenses.

DESCRIPTION OF THE INVENTION

Free radicals have come to be appreciated for their importance to humanhealth and disease. Many common and life-threatening diseases, includingatherosclerosis, cancer, and aging, have free radical reactions as anunderlying mechanism of injury. One of the most common types of radicalsis the reactive oxygen species (ROS). These are the products of normalcell respiration and metabolism and are generally regulated by cellulardefense systems present in the body. Such cellular defense systemsreduce the amount of damage that free and reactive species radicals maycause by scavenging free radicals or enzymatically converting the freeradicals to less toxic chemical species, thereby serving a physiologicalrole as antioxidants.

Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase(GPX) are three mammalian enzymes important to cellular defense againstROS-mediated cellular damage. Due to environmental agents such aspollution, and lifestyle factors such as smoking or exercising, thecellular exposure to free radicals is increased. Such increase may bringthe body out of balance, especially as the body ages and the mechanismsthat produce antioxidants or remove ROS are insufficient to counteroxidative stress. The body's antioxidant defense system(s), includingSOD, CAT, and GPX, can be impaired by the aging process and orcompromised, for example, by inflammation, microbial infection, viralinfection, the progression of cancer or neurological disorders, and manyother disorders characterized by or caused in part by oxidative stress.The resulting damage can range from disruption of biological processes,killing of cells, and mutation of genetic material, which may lead tothe occurrence of cancer. Accordingly, the potential use of antioxidantcontaining dietary supplements for protection against the effects ofoxidative stress and the progression of degenerative diseases and aginghas been the subject of an increasing number of studies during the pastfour decades, see for example, Pauling L., N Engl J Med., Vitamin Ctherapy of advanced cancer, Mar. 20, 1980; 302(12):694-5.

Non-enzymatic antioxidants can react with free radicals directly andbecome self-oxidized (therefore no longer available to quench freeradicals); or one antioxidant may act as a reducing agent and anotherantioxidant oxidized in cyclical fashion (e.g., the interaction ofascorbic acid and alpha-tocopherol). Other non-enzymatic free radicalscavengers have been used experimentally with varying results (e.g.mannitol, PBS, etc.); their clinical use is severely limited due totheir toxicities. Other synthetic antioxidants, e.g., BHA (butylatedhydroxy anisole), BHT (butylated hydroxy toluene) and NDGA (nordihydroguaiaretic acid) may cause allergic reactions and oncogenesis due totheir strong toxicity in the body, and be easily disrupted by heat dueto temperature sensitivity.

Enzymatic antioxidants (e.g., SOD, CAT and GPX), on the other hand, arenot consumed in the reactions with free radicals, although they can bedamaged under pathological conditions and consequently renderednon-functional. In the local cellular milieu, damaged enzymaticantioxidants would render that cellular environment compromised andsubject to free radical attack. The disadvantage of administeringenzymatic antioxidants to humans is (1) the possibility of allergicreactions (in the case of a bacterial or fungal derived enzyme) ofvarying degrees of severity; (2) the great cost of harvesting theseenzymes; (3) the limitation of quantities of enzymatic antioxidants ableto be administered at a given time (theoretically to avoid side-effectssuch as serum sickness or other immune reaction to the recombinantprotein); (4) they serve a singular purpose (i.e., they react with onlyone type of oxidant); and (5) they do not quench all radicals, that maybe important for beneficial metabolic pathways, e.g., nitric oxideinduced vasodilation and immune system support (see, e.g., Griscavage JM, Wilk S, Ignarro L J., Proc Natl Acad Sci USA., Inhibitors of theproteasome pathway interfere with induction of nitric oxide synthase inmacrophages by blocking activation of transcription factor NF-kappa B.,Apr. 16, 1996; 93(8):3308-12.

The efficacy of direct oral administration of SOD, CAT, or GPX has beenlimited by the sensitivity of these enzymes to the milieu of thedigestive system and/or lack of bioavailability. For example, researchindicated that the digestive system destroyed SOD, and that neither CATnor GPX was absorbed via the digestive tract.

Since administration by direct ingestion of the antioxidants showeddisappointing results, efforts have been directed to provide the bodywith the so-called “building blocks” of each of the three antioxidants.A promising approach has been the development of compositions directedtoward increasing the activities of SOD, CAT, and GPX in the body.Accordingly, supplements have been formulated to increase levels of thebody's zinc, copper, and manganese, to assist the body's production ofSOD. Similarly, iron, selenium, and glutathione related supplements havebeen developed to increase CAT and GPX. These compounds have toxiceffects in large quantities.

SUMMARY OF THE INVENTION

The present invention provides compositions that can be administered toa mammalian subject, and will safely induce in the subject, increasedantioxidant potential in the subject by increasing the activity of atleast one antioxidant enzyme which include, e.g., SOD, CAT, and GPX, andthereby decreasing the tissue level of pathologic free radical species.The compositions of the present invention yield an overall net decreasein oxidative stress and inflammation when administered to a mammaliansubject in an effective amount with minimal undesirable side-effects.Advantageously, the compositions of the invention provides fewer sideeffects than may be associated with each of the individual active agentsin the composition.

In one aspect, the invention provides an antioxidant-promotingcomposition that comprises active ingredients comprising at least twoagents that include, e.g., Bacopa monniera extract; milk thistleextract, ashwagandha powder, green tea extract, Gotu kola powder, Ginkobiloba leaf extract; Aloe vera powder; turmeric extract; and N-acetylcysteine, wherein the composition increases the enzyme activity level ofat least one antioxidant enzyme, e.g., superoxide dismutase; catalase;and glutathione peroxidase and decreases the plasma concentration levelof thiobarbituric acid reactive chemical species, when administered inan effective amount to a mammalian subject in need thereof. In oneembodiment, the Bacopa monniera extract is standardized extract preparedfrom the leaves of the Bacopa monniera plant. In one embodiment, theBacopa monniera standardized extract contains at least 20% bacosides. Inone embodiment, the Bacopa monniera extract is present at aconcentration of about 5 weight percent to about 50 weight percent ofthe total dry weight of active ingredients of the composition. In oneembodiment, the Bacopa monniera extract is present at a concentration ofabout 10 weight percent to about 30 weight percent of the total dryweight of active ingredients of the composition. In one embodiment, theBacopa monniera extract is present at a concentration at least about 22weight percent of the total dry weight of active ingredients of thecomposition. In one embodiment, Bacopa monniera extract is present at aconcentration at least about 12 weight percent of the total dry weightof active ingredients of the composition. In one embodiment, the milkthistle extract is standardized to contain at least about 70 weightpercent silymarin. In one embodiment, the milk thistle extract ispresent at a concentration from about 5 weight percent to about 60weight percent of the total dry weight of active ingredients of thecomposition. In one embodiment, the milk thistle extract is present at aconcentration from about 10 weight percent to about 50 weight percent ofthe total dry weight of active ingredients of the composition. In oneembodiment, the milk thistle extract is present at a concentration atleast about 33 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the milk thistleextract is present at a concentration at least about 22 weight percentof the total dry weight of active ingredients of the composition. In oneembodiment, the ashwagandha powder is present at a concentration fromabout 5 weight percent to about 50 weight percent of the total dryweight of active ingredients of the composition. In one embodiment, theashwagandha powder is present at a concentration from about 10 weightpercent to about 30 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the ashwagandhapowder is present at a concentration at least about 22 weight percent ofthe total dry weight of active ingredients of the composition. In oneembodiment, the ashwagandha powder is present at a concentration atleast about 12 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the turmeric extractis standardized to contain at least about 95 weight percent curcumin. Inone embodiment, turmeric extract is present at a concentration fromabout 2.5 weight percent to about 25 weight percent of the total dryweight of active ingredients of the composition. In one embodiment, theturmeric extract is present at a concentration from about 5 weightpercent to about 15 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the turmeric extractis present at a concentration at least about 11 weight percent of thetotal dry weight of active ingredients of the composition. In oneembodiment, the turmeric extract is present at a concentration at leastabout 6 weight percent of the total dry weight of active ingredients ofthe composition. In one embodiment, the green tea extract isstandardized to contain at least about 40% polyphenols. In oneembodiment, the at least one polyphenol is polyphenol(−)-epigallocatechin gallate. In one embodiment, the green tea extractis present at a concentration from about 2.5 weight percent to about 25weight percent of the total dry weight of active ingredients of thecomposition. In one embodiment, the green tea extract is present at aconcentration from about 5 weight percent to about 15 weight percent ofthe total dry weight of active ingredients of the composition. In oneembodiment, the green tea extract is present at a concentration at leastabout 11 weight percent of the total dry weight of active ingredients ofthe composition. In one embodiment, the green tea extract is present ata concentration at least about 6 weight percent of the total dry weightof active ingredients of the composition. In one embodiment, the Gotukola powder is present at a concentration from at least about 5 weightpercent to about 50 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the Gotu kola powderis present at a concentration from at least about 10 weight percent toabout 30 weight percent of the total dry weight of active ingredients ofthe composition. In one embodiment, the Gotu kola extract is present ata concentration at least about 12 weight percent of the total dry weightof active ingredients of the composition. In one embodiment, the Ginkobiloba leaf extract is present at a concentration from at least about 5weight percent to about 50 weight percent of the total dry weight ofactive ingredients of the composition. In one embodiment, the Ginkobiloba leaf extract is present at a concentration from at least about 10weight percent to about 30 weight percent of the total dry weight ofactive ingredients of the composition. In one embodiment, the Ginkobiloba leaf extract is present at a concentration at least about 12weight percent of the total dry weight of active ingredients of thecomposition. In one embodiment, the Aloe vera powder is present at aconcentration from at least about 5 weight percent to about 50 weightpercent of the total dry weight of active ingredients of thecomposition. In one embodiment, the Aloe vera powder is present at aconcentration from at least about 10 weight percent to about 30 weightpercent of the total dry weight of active ingredients of thecomposition. In one embodiment, the Aloe vera powder is present at aconcentration at least about 12 weight percent of the total dry weightof active ingredients of the composition. In one embodiment, theN-acetyl cysteine is present at a concentration of about 2 weightpercent to about 20 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the N-acetyl cyteineis present at a concentration of about 5 weight percent to about 15weight percent of the total dry weight of active ingredients of thecomposition. In one embodiment, the N-acety cysteine is present at aconcentration of at least about 6 weight percent of the total dry weightof active ingredients of the composition.

In one embodiment, the composition of the invention contains at leasttwo (2) of the active ingredients of the antioxidant-promotingcomposition. In one embodiment, the composition of the inventioncontains at least three (3) of the active ingredients of theantioxidant-promoting composition. In one embodiment, the composition ofthe invention contains at least four (4) of the active ingredients ofthe antioxidant-promoting composition. In one embodiment, thecomposition of the invention contains at least five (5) of the activeingredients of the antioxidant-promoting composition. In one embodiment,the composition of the invention contains at least six (6) of the activeingredients of the antioxidant-promoting composition. In one embodiment,the composition of the invention contains at least seven (7) of theactive ingredients of the antioxidant-promoting composition. In oneembodiment, the composition of the invention contains at least eight (8)of the active ingredients of the antioxidant-promoting composition. Inone embodiment, the composition of the invention contains at least nine(9) of the active ingredients of the antioxidant-promoting composition.

In another embodiment, the composition of the invention is anantioxidant-promoting composition comprising active ingredientscomprising Bacopa monniera extract; milk thistle extract, ashwagandhapowder, green tea extract, and turmeric extract, wherein the compositionincreases the enzyme activity level of at least one antioxidant enzyme,e.g., superoxide dismutase; catalase; and glutathione peroxidase anddecreases the plasma concentration level of thiobarbituric acid reactivechemical species, when administered in an effective amount to amammalian subject in need thereof. In one embodiment, the Bacopamonniera extract is a standardized extract prepared from the leaves ofthe Bacopa monniera plant. In one embodiment, the standardized Bacopamonniera extract contains at least 20% bacosides. In one embodiment, theBacopa monniera extract is present at a concentration of about 5 weightpercent to about 50 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the Bacopa monnieraextract is present at a concentration of about 10 weight percent toabout 30 weight percent of the total dry weight of active ingredients ofthe composition. In one embodiment, the Bacopa monniera extract ispresent at a concentration at least about 22 weight percent of the totaldry weight of active ingredients of the composition. In one embodiment,the Bacopa monniera extract is present at a concentration at least about12 weight percent of the total dry weight of active ingredients of thecomposition. In one embodiment, the milk thistle extract is standardizedto contain at least about 70 weight percent silymarin. In oneembodiment, the milk thistle extract is present at a concentration fromabout 5 weight percent to about 60 weight percent of the total dryweight of active ingredients of the composition. In one embodiment, themilk thistle extract is present at a concentration from about 10 weightpercent to about 50 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the milk thistleextract is present at a concentration at least about 33 weight percentof the total dry weight of active ingredients of the composition. In oneembodiment, the milk thistle extract is present at a concentration atleast about 22 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the ashwagandhapowder is present at a concentration from about 5 weight percent toabout 50 weight percent of the total dry weight of active ingredients ofthe composition. In one embodiment, the ashwagandha powder is present ata concentration from about 10 weight percent to about 30 weight percentof the total dry weight of active ingredients of the composition. In oneembodiment, the ashwagandha powder is present at a concentration atleast about 22 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the ashwagandhapowder is present at a concentration at least about 12 weight percent ofthe total dry weight of active ingredients of the composition. In oneembodiment, the turmeric extract is standardized to contain at leastabout 95 weight percent curcumin. In one embodiment, the turmericextract is present at a concentration from about 2.5 weight percent toabout 25 weight percent of the total dry weight of active ingredients ofthe composition. In one embodiment, the turmeric extract is present at aconcentration from about 5 weight percent to about 15 weight percent ofthe total dry weight of active ingredients of the composition. In oneembodiment, the turmeric extract is present at a concentration at leastabout 11 weight percent of the total dry weight of active ingredients ofthe composition. In one embodiment, the turmeric extract is present at aconcentration at least about 6 weight percent of the total dry weight ofactive ingredients of the composition. In one embodiment, the green teaextract is standardized to contain at least about 40% polyphenols. Inone embodiment, at least one polyphenol is polyphenol(−)-epigallocatechin gallate. In one embodiment, the green tea extractis present at a concentration from about 2.5 weight percent to about 25weight percent of the total dry weight of active ingredients of thecomposition. In one embodiment, the green tea extract is present at aconcentration from about 5 weight percent to about 15 weight percent ofthe total dry weight of active ingredients of the composition. In oneembodiment, the green tea extract is present at a concentration at leastabout 11 weight percent of the total dry weight of active ingredients ofthe composition. In one embodiment, the green tea extract is present ata concentration at least about 6 weight percent of the total dry weightof active ingredients of the composition. In one embodiment, thecomposition further comprises the active ingredients Gotu kola powder,Ginko biloba leaf extract and Aloe vera powder. In one embodiment, theGotu kola powder is present at a concentration from at least about 5weight percent to about 50 weight percent of the total dry weight ofactive ingredients of the composition. In one embodiment, the Gotu kolapowder is present at a concentration from at least about 10 weightpercent to about 30 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the Gotu kola powderextract is present at a concentration at least about 12 weight percentof the total dry weight of active ingredients of the composition. In oneembodiment, the Ginko biloba leaf extract is present at a concentrationfrom at least about 5 weight percent to about 50 weight percent of thetotal dry weight of active ingredients of the composition. In oneembodiment, the Ginko biloba leaf extract is present at a concentrationfrom at least about 10 weight percent to about 30 weight percent of thetotal dry weight of active ingredients of the composition. In oneembodiment, the Ginko biloba leaf extract is present at a concentrationat least about 12 weight percent of the total dry weight of activeingredients of the composition. In one embodiment, the Aloe vera powderis present at a concentration from at least about 5 weight percent toabout 50 weight percent of the total dry weight of active ingredients ofthe composition. In one embodiment, the Aloe vera powder is present at aconcentration from at least about 10 weight percent to about 30 weightpercent of the total dry weight of active ingredients of thecomposition. In one embodiment, the Aloe vera powder is present at aconcentration at least about 12 weight percent of the total dry weightof active ingredients of the composition.

In some embodiments, the compositions of the present invention furthercomprise at least one excipient, e.g., calcium carbonate; croscarmellosesodium; dicalcium phosphate; magnesium stearate; microcrystallinecellulose; modified cellulose; silica; and stearic acid.

In another embodiment the invention provides an antioxidant-promotingcomposition comprising: (a) active ingredients comprising at least about150 milligrams Bacopa monniera, extract 45 percent bacosides; at leastabout 225 milligrams milk thistle extract, between about 70 percent andabout 80 percent silymarin; at least about 150 mg ashwagandha powder; atleast about 75 milligrams green tea extract; 98 percent polyphenols; 45percent (−)-epigallocatechin gallate; at least about 75 milligramsturmeric extract; 95 percent curcumin; and (b) other ingredientscomprising calcium carbonate; croscarmellose sodium; dicalciumphosphate; magnesium stearate; microcrystalline cellulose; modifiedcellulose; silica; and stearic acid; wherein the composition increasesthe enzyme activity level of at least one antioxidant enzyme selectedfrom the group consisting of: superoxide dismutase; catalase; andglutathione peroxidase and decreases the plasma concentration level ofthiobarbituric acid reactive chemical species, when administered in aneffective amount to a mammalian subject in need thereof. In oneembodiment, the composition is formulated as an oral dosage form. In oneembodiment, the oral dosage form is selected from the group consistingof: a tablet; capsule; and caplet.

In another aspect the invention provides a method of increasing theantioxidant activity level of a mammalian subject in need thereof, byincreasing the level of enzyme activity of at least one enzyme, e.g.,superoxide dismutase; catalase; and glutathione peroxidase, byadministering to the subject an effective amount of anantioxidant-promoting composition of the invention, wherein theincreased enzyme activity decreases the tissue damage caused bypathological free radicals. In one embodiment, the tissue damage causedby pathological free radicals occurs in a mammalian subject with adisease or condition selected from the group which includes, e.g.,inflammation; infection; atherosclerosis; hypertension; cancer;radiation injury; neurological disease; neurodegenerative disease;ischemia/reperfusion injury; aging; wound healing; glutathionedeficiency; acquired immunodeficiency syndrome; sickle cell anemia; anddiabetes mellitus. In one embodiment of the method, theantioxidant-promoting composition is administered as an oral dietarysupplement.

In one embodiment, the invention provides a method of reducing theplasma level of c-reactive protein in a mammalian subject, byadministering to the subject an effective amount of anantioxidant-promoting composition of the invention. In one embodiment ofthe method, the antioxidant-promoting composition is administered as anoral dietary supplement. In one embodiment of the method, the plasmalevel of c-reactive protein decreases by at least 10% or more in a 30day period. In one embodiment of the method, the plasma level ofc-reactive protein decreases by at least 20% or more in a 30 day period.

In one embodiment, the invention provides a method of reducing the bloodpressure in a mammalian subject, by administering to the subject aneffective amount of an antioxidant-promoting composition of theinvention. In one embodiment of the method, the antioxidant-promotingcomposition is administered as an oral dietary supplement.

The compositions of the present invention are useful to promotehealthful benefits as follows: relaxation; bone marrow and women'shealth (e.g., stabilizes fetus and regenerates hormones); mentalfunction (e.g., memory and concentration); sexual function; diuretic;anti-inflammatory; anti-mutagenic agent; anti-cancer agent;cholagogueue; depurative; fumitory; hemostatic agent; hepatoprotectiveagent; lactagogue; stomachic; tonic; vulnerary; tissue healing (e.g.;wounds; skin; other connective tissues; lymph tissue; blood vessels; andmucous membranes); remove free radicals in the peripheral and/orcerebral vascular systems; and inhibit lipid peroxidation; improvecerebral blood circulation and to protect the nerves against damagingfree radicals; protective against cell damage caused by chemotherapy andradiation therapy; to enhance immune function; and to protect againsttoxins (e.g., acetametaphen and other drugs; mercury; lead).

The compositions of the present invention are useful to prevent or treatthe following disorders and diseases: memory loss; Parkinson's disease;aging; toxin-induced hepatotoxicity; inflammation; liver cirrhosis;chronic hepatitis; and diabetes due to cirrhosis; indigestion; fatigue;stress; cough; infertility; tissue inflammation; cancer; anxietydisorders; panic attacks; rheumatism; pain; manic depression; alcoholicparanoia; schizophrenia; fever; insomnia; infertility; aging; skininflammations and disorders; alcoholism; anemia; carbuncles;convalescence; emaciation; HIV; AIDS; immune system problems; lumbago;multiple sclerosis; muscle energy loss; paralysis; swollen glands;ulcers; breathing difficulties; inflammation; psoriasis; cancer (e.g.;prostate cancer, lung cancer and breast cancer); pain; cardiovasculardisease (e.g.; arteriosclerosis and atherosclerosis);ischemia/reperfusion injury; anxiety; attention deficit disorder;leprosy; arthritis (e.g., psoriatic arthritis; anklylosing spondvlitis;and rheumatoid arthritis); hemorrhoids; tuberculosis; high bloodpressure; congestive heart failure; venous insufficiency (pooling ofblood in the veins; usually in the legs); sore throat; hepatitis;syphilis; stomach ulcers; epilepsy; diarrhea; asthma; burns; piles;sunburn; wrinkles; headache; insect bites; cuts; ulcers; sores; herpes;jaundice; bursitis; canker sores; sore gums; poison ivy; gastritis; highcholesterol; heart disease; bacterial infection; viral infection; acne;aging; immune disorders; dental caries; periodontitis; halitosis;dandruff; cardiovascular disease (e.g., hypertension; thrombosis;arteriosclerosis); migraine headaches, diabetes; elevated blood glucose;diseases of the alimentary canal and respiratory system; age-relatedphysical and mental deterioration (e.g., Alzheimer's Disease andage-related dementia); cardiovascular disease; cerebral vascularinsufficiency and impaired cerebral performance; congestive symptoms ofpremenstrual syndrome; allergies; age-related vision loss; depression;Raynaud's disease; peripheral vascular disease; intermittentclaudication; vertigo; equilibrium disorder; prevention of altitudesickness; tinnitus (ringing in the ear); liver fibrosis; maculardegeneration; asthma; graft rejection; and immune disorders that inducetoxic shock; bronchoulmonary disease as cystic fibrosis; chronicbronchitis; gastritis; heart attack; angina pectoris; chronicobstructive pulmonary disease; kidney damage during coronaryangiography; Unverricht-Lundborg disease; pseudoporphyria; pneumonia;and paracetamol hepatotoxicity

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating effect of Protandim I dietarysupplementation on percent weight gain of mice after 23 days.

FIG. 2 shows graphs illustrating effect of Protandim I dietarysupplement on murine red blood cell SOD concentration (RBC SOD). Panel Ashows a graph of RBC SOD concentration (U/ml) observed in mice fed 1×,3× and 10× dosage of Protandim I dietary supplement for 23 days. Panel Bshows a graph of the percent increase in RBC SOD after 23 days on 1×, 3×and 10× dosage of Protandim I dietary supplement. An asterisk indicatedstatistical significance at p<0.02 level.

FIG. 3 shows graphs effect of Protandim I dietary supplement on murineliver SOD concentration. Panel A shows a graph of liver SODconcentration (U/mg protein) observed in mice fed for 23 days on 1×, 3×and 10× dosage of Protandim I dietary supplement. Panel B shows a graphof the percent increase in liver SOD after 23 days on 1×, 3× and 10×dosage of Protandim I dietary supplement. An asterisk indicatesstatistical significance at p<0.02 level. A double asterisk indicatesstatistical significance at p<0.0001 level.

FIG. 4 shows graphs illustrating effect of Protandim I dietarysupplement on murine brain SOD concentration. Panel A shows a graph ofbrain SOD concentration (U/mg protein) observed in mice fed 1×, 3× and10× dosage of Protandim I dietary supplement for 23 days. Panel B showsa graph of the percent change in brain SOD after 23 days on 1×, 3× and10× dosage of Protandim I dietary supplement. An asterisk indicatesstatistical significance at p<0.02 level.

FIG. 5 shows graphs illustrating effect of Protandim I dietarysupplement on murine red blood cell catalase concentration (RBC CAT).Panel A shows a graph of RBC CAT concentration (U/ml) observed in micefed 1×, 3× and 10× dosage of Protandim I dietary supplement for 23 days.Panel B shows a graph of the percent increase in RBC CAT after 23 dayson 1×, 3× and 10× dosage of Protandim I dietary supplement.

FIG. 6 is a graph illustrating effect of Protandim I dietary supplementon murine liver CAT concentration (U/mg protein) observed in mice fed1×, 3× and 10× dosage of Protandim I dietary supplement for 23 days.

FIG. 7 shows graphs effect of Protandim I dietary supplement on murinered blood cell glutathione peroxidase concentration (RBC GPX). Panel Ashows a graph of RBC GPX concentration (U/ml) observed in mice fed 1×,3× and 10× dosage of Protandim I dietary supplement for 23 days. Panel Bshows a graph of the percent change in RBC GPX (U/g Hb) after 23 days on1×, 3× and 10× dosage of Protandim I dietary supplement.

FIG. 8 shows graphs illustrating effect of Protandim I dietarysupplement on murine liver GPX concentration. Panel A shows a graph ofliver GPX concentration (U/mg protein) observed in mice fed 1×, 3× and10× dosage of Protandim I dietary supplement for 23 days. Panel B showsa graph of the percent change in liver GPX after 23 days on 1×, 3× and10× dosage of Protandim I dietary supplement. An asterisk indicatedstatistical significance at p<0.004 level.

FIG. 9 shows graphs illustrating effect of Protandim I dietarysupplement on murine brain GPX concentration. Panel A shows a graph ofbrain GPX concentration (U/mg protein) observed in mice fed 1×, 3× and10× dosage of Protandim I dietary supplement for 23 days. Panel B showsa graph of the percent change in brain GPX after 23 days on variousdiets of a composition of the present invention. An asterisk indicatesstatistical significance at p<0.03 level.

FIG. 10 shows graphs illustrating effect of Protandim I dietarysupplement on murine plasma lipid peroxidation products measured asTBARS. Panel A shows a graph of plasma TBARS concentration (μM) observedin mice fed 1×, 3× and 10× dosage of Protandim I dietary supplement for23 days. Panel B shows a graph of the percent change in plasma TBARSafter 23 days on 1×, 3× and 10× dosage of Protandim I dietarysupplement. A single asterisk indicates statistical significance atp<0.004 level. A double asterisk indicates statistical significance atthe p<0.0004 level.

FIG. 11 shows graphs illustrating the effect of diets of a compositionof the present invention on murine liver lipid peroxidation productsmeasured as TBARS. Panel A shows a graph of liver TBARS concentration(nmol/mg protein) observed in mice fed 1×, 3× and 10× dosage ofProtandim I dietary supplement for 23 days. Panel B shows a graph of thepercent change in liver TBARS after 23 days on 1×, 3× and 10× dosage ofProtandim I dietary supplement. A single asterisk indicates statisticalsignificance at p=0.001 level. A double asterisk indicates statisticalsignificance at the p<0.00001 level.

FIG. 12 shows graphs illustrating effect of Protandim I dietarysupplement on murine brain lipid peroxidation products measured asTBARS. Panel A shows a graph of brain TBARS concentration (nmol/mgprotein) observed in mice fed 1×, 3× and 10× dosage of Protandim Idietary supplement for 23 days. Panel B shows a graph of the percentchange in brain TBARS after 23 days 1×, 3× and 10× dosage of Protandim Idietary supplement. A single asterisk indicates statistical significanceat p<0.004 level. A double asterisk indicates statistical significanceat the p<0.0001 level.

FIG. 13 shows graphs illustrating effect of Protandim I dietarysupplement on murine SOD, CAT and GPX in various tissues. Panel A showsa graph of the percent change in SOD observed in RBC, liver and brain ofmice fed 1×, 3× and 10× dosage of Protandim I dietary supplement for 23days. Panel B shows a graph of the percent change in CAT observed in RBCand liver of mice fed 1×, 3× and 10× dosage of Protandim I dietarysupplement for 23 days. Panel C shows a graph of the percent change inGPX observed in RBC, liver and brain of mice fed 1×, 3× and 10× dosageof Protandim I dietary supplement for 23 days. A single asteriskindicates statistical significance at relative to the control diet.

FIG. 14 is a graph illustrating the effect of Protandim I dietarysupplement on murine lipid peroxidation in various tissues. The graphshows the percent change in lipid peroxidation measured as TBARS in RBC,liver and brain of mice fed 1×, 3× and 10× dosage of Protandim I dietarysupplement for 23 days. A single asterisk indicates statisticalsignificance at relative to the control diet.

FIG. 15 is a graph showing response of human subjects to dietarysupplementation with the herbal composition Protandim II. Panel A showsplasma TBARS level in human subjects prior to supplementation withProtandim II at 675 mg/day (closed circles), after 30 days ofsupplementation (gray squares), and after 120 days (open circles). Thelevels of plasma TBARS dropped an average of 51% (p<0.002) after 30 daysof Protandim treatments (gray squares) the age-related increase in TBARSvirtually disappeared. Panel B shows plasma TBARS level in normalsubjects before supplementation with Protandim II showed a strongage-dependent increase in TBARS (circles). The levels of plasma TBARSdropped on average 51% (p<0.002) after 30 days of Protandim II (squares)supplementation, and the age-related increase in TBARS virtuallydisappeared.

FIG. 16 is a graph illustrating the effect of Protandim II dietarysupplementation on human SOD in RBCs.

FIG. 17 is a graph illustrating the effect of Protandim II dietarysupplementation on human CAT in RBCs.

FIG. 18 is a graph illustrating the effect of Protandim II dietarysupplementation on human CRP protein level.

FIG. 19 is a graph illustrating the effect of half-dose Protandim II onplasma TBARS in humans subjects. The half-dose Protandim II was 338mg/day dietary supplement.

FIG. 20 is a graph comparing the effect of Protandim II dosage on plasmaTBARS in human subjects. Average plasma TBARS concentrations are shownafter 30 days supplementation of Protandim II at full-dose (675 mg/day;n=11) and half-dose (338 mg/day; n=4). The difference was statisticallysignificant at p<0.03 as determined by one-tailed paired t-test.

FIG. 21 shows graphs detailing the blood pressure measurements of humansubjects administered an herbal composition of the invention. Panel Ashows the blood pressure measurements for Subject #2 in Example 2. PanelB shows the blood pressure measurements for Subject #4 in Example 2. Thearrow indicates the point in time at which the individual begantreatment with an herbal composition of the invention. In bothindividuals, diastolic blood pressure dropped significantly over aperiod of about 21 days, remaining constant thereafter.

DETAILED DESCRIPTION OF THE INVENTION

There remains a need to formulate improved compositions and supplementsthat increase the SOD, CAT, or GPX levels in a mammal, that may be moreeffective and have fewer side-effects and lowered toxicity. The presentinvention provides compositions that can be administered to a mammaliansubject, and will safely induce in the subject, increased antioxidantpotential in the subject by increasing the activity of at least oneantioxidant enzyme which include, e.g., SOD, CAT, and GPX, and therebydecreasing the tissue level of pathologic free radical species. Thecomposition provides for an overall net decrease in oxidative stressexperienced by the mammal, with minimal undesirable side-effects.Advantageously, the composition itself provides fewer side effects thanmay be associated with each of the individual active agents in thecomposition.

Accordingly, the various aspects of the present invention relate totherapeutic or prophylactic uses of certain particular compositions(e.g., dietary supplement compositions) in order to prevent or treat adisease or an injury induced by pathological free and reactive radicalreactions. The various aspects of the present invention further relateto therapeutic or prophylactic uses of certain particular compositionsin order to prevent or treat a disease or an injury associated withdecreased SOD, CAT, and/or GPX enzyme activity, or that would benefitfrom increased SOD, CAT, and/or GPX enzyme activity, such asinflammation and oxidative stress.

Various particular embodiments that illustrate these aspects follow.

It is to be appreciated that the various modes of treatment orprevention of medical conditions as described are intended to mean“substantial”, which includes total but also less than total treatmentor prevention, and wherein some biologically or medically relevantresult is achieved. A “subject,” as used herein, is preferably a mammal,such as a human, but can also be an animal, e.g., domestic animals(e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs,horses and the like) and laboratory animals (e.g., rats, mice, guineapigs and the like). An “effective amount” of an antioxidant inducingcomposition, as used herein, is a quantity of the composition providedby a particular route of administration and at a particular dosingregimen, that is sufficient to achieve a desired therapeutic and/orprophylactic effect. For example, an amount that results in theprevention of or a decrease in the symptoms associated with a diseasethat is being treated. The amount of the composition administered to thesubject will depend on the type and severity of the disease, theamenability of the disorder to respond to antioxidants, and on thecharacteristics of the individual and their metabolic ability to respondto the compositions to produce in vivo, the antioxidants, such factorsincluding general health, age, sex, body weight and tolerance to theactive agents in the compositions. The skilled artisan will be able todetermine appropriate dosages depending on these and other factors.Typically, an effective amount of the compositions of the presentinvention, sufficient for achieving a therapeutic or prophylacticeffect, range from about 0.000001 mg per kilogram body weight per day toabout 10,000 mg per kilogram body weight per day. Preferably, the dosageranges are from about 0.0001 mg per kilogram body weight per day toabout 100 mg per kilogram body weight per day. The compositions of thepresent invention can also be administered in combination with eachother, or with one or more additional therapeutic or prophylacticcompounds.

It is advantageous to formulate oral compositions in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the dietary supplement and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. Typically, anoral dose is taken two-times to four-times daily, until symptom reliefis apparent. The compositions of the present invention can also beadministered in combination with each other, or with one or moreadditional therapeutic compounds.

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent invention. In general, such disclosure provides compositionsbeneficial to the health of a subject. In one embodiment, thecompositions of the invention is a dietary supplement composition. Inanother embodiment, the compositions of the invention are combined withother dietary supplement compositions. In yet other embodiments, thecomposition of the invention is a pharmaceutical formulation.

Radicals, Oxidative Stress and Cellular Defense

Over the past few decades, free and bound reactive radicals, highlyreactive and thereby destructive molecules, have come to be appreciatedincreasingly for their importance to human health and disease. Manycommon and life-threatening human diseases, including atherosclerosis,cancer, and aging, have radical-based pathological reactions as anunderlying mechanism of injury.

A radical, or a free radical, is generally understood as a molecule withone or more unpaired electrons in its outer orbital shell. Manymolecular species with bound radicals are monoxides or other oxygencontaining compounds, generally referred to as reactive oxygen species(ROS). These highly unstable molecules tend to react rapidly withadjacent molecules, donating, abstracting, or even sharing their outerorbital electron(s). This reaction not only changes the adjacent, targetmolecule, sometimes in profound and beneficial ways, but it can alsodamage it, or alternatively the unpaired electron can be passed along tothe target, i.e., as in a free radical, generating a second unwantedROS, which can then go on to react positively or detrimentally with anew target. In fact, much of the high reactivity of ROS is due to theirgeneration of such molecular chain reactions, effectively amplifyingtheir effects many fold. Antioxidants afford protection because they canscavenge ROS and free radicals before they cause damage to the variousbiological molecules, or prevent oxidative damage from spreading, e.g.,by interrupting the radical chain reaction of lipid peroxidation. Thereactivity of radicals in the body, and the burden on the body thatresults in eventual pathological conditions, is known as oxidativestress.

For example, oxidative stress in the circulatory system is seen asatherosclerosis and by plaque deposition on the vessel walls. Plaquescause inflammatory responses, which increase radical formation byrecruited immune cells, which worsens the cycle. It has been observedthat the prime targets of both free radicals and ROS are thepolyunsaturated fats in the membrane lipids of cells. The oxidativedeterioration of polyunsaturated fats is known as lipid peroxidation.Lipid peroxidation severely impairs membrane function, which is believedto lead to the disorganization of cell structure and function. Productsof lipid peroxidation such as malondialdehyde, a known mutagen reactivewith proteins and amino acids, are a good measure of the amount ofoxidative stress on the body. Lipofuscin, another byproduct of lipidperoxidation, accumulates in the body with age and it is believed thatcytosolic buildup of this byproduct compromises brain function. Whatbegins as localized pathological radical reactions thus result inoxidative stress that can and will eventually impact distal organsystems.

Vitamins such as vitamin C and vitamin E, both of which are found infoods and available as supplements, help the body reduce effects ofoxidative stress. A more powerful combatant against the free radicalsand ROS, however, is the body's own self defense system of naturallyproduced chemicals called antioxidants. These antioxidants act toterminate the propagation of free and bound radicals on ROS either bygiving an electron to the free radical or ROS or by hindering theirformation.

The body's antioxidant defense system includes three important naturalantioxidants: superoxide dismutase (SOD), catalase (CAT), andglutathione peroxidase (GPX). Studies conducted have indicated thatthese antioxidants can work synergistically as the reactions theycatalyze are metabolically sequential, beginning first with SOD followedby the actions of CAT and GPX.

SODs are a class of enzymes that catalyze reactions similar to the onebelow:O2-+O2-+2H+→H2O2+O2

The role of SOD in the defense system is to remove superoxide radicals,which are a type of ROS. SOD is found in the body, primarily in threeforms: (1) in the cytoplasm as Cu—Zn SOD; (2) in the mitochondrion asMn-SOD; (3) and in an extracellular environment as CuSOD. Accordingly,the body is dependent upon the presence of zinc, copper, and manganesefor the manufacture of SOD. During the removal of superoxide radicals bySOD, both hydroxyl and oxygen radicals are produced, which are catalyzedby CAT and GPX respectively.

Catalase (CAT) is present in the peroxisomes of nearly all aerobiccells, and serves to protect the cell from the toxic effects of hydrogenperoxide by catalyzing its decomposition into molecular oxygen and waterwithout the production of free radicals.

The overall reaction is as follows:2H2O2→2H2O+O2

The protein exists as a dumbbell-shaped tetramer of four identicalsubunits (220,000 to 350,000 kDa). Each monomer contains a hemeprosthetic group at the catalytic center. CAT monomers from certainspecies (e.g., cow) also contain one tightly bound NADP per subunit.This NADP may serve to protect the enzyme from oxidation by its H2O2substrate. The body's production of CAT is dependent upon theavailability of iron.

Glutathione peroxidases (GPXs) are a large class of diverse enzymeswhich catalyze the reduction of hydrogen peroxide (H2O2), organichydroperoxides and lipid hydroperoxides to the corresponding alcoholusing glutathione (GSH; glutamylcysteinylglycine) as the electron donor(Ursini et al., 1995). The GPX enzymes catalyze the reduction of H2O2 towater and organic peroxides (R—O—O—H) to the corresponding stablealcohols (R—O—H) using glutathione (GSH) as a source of reducingequivalents:2GSH→ROH+GSSG+H2O

GPXs are important in helping to protect cells against oxidative damage,particularly lipid peroxidation. (Flohé et al., 1976). In mammals, thecytosolic (c) GPX family as well as a family of phospholipidhydroperoxide (PH) GPXs possess a selenocysteine residue at their activesite. With the exception of phospholipid-hydroperoxide GPX, a monomer,all of the GPX enzymes are comprised of four identical subunits (monomerMr 22-23 kDa). Each subunit contains a molecule of selenocysteine in theenzyme active site. The selenocysteine is thought to participatedirectly in electron donation to the peroxide substrate and becomeoxidized in the process. The enzyme then uses glutathione as an electrondonor to regenerate the reduced form of the selenocysteine (Ursini,1995). The GPX enzymes accept a wide variety of organic peroxides assubstrates. The body's ability to produce GPX is in part dependent uponthe adequate supply of selenium, and the supply of glutathione, atripeptide that the body produces from the amino acids cysteine,glutamic acid, and glycine. Mammals contain plasma (p) GPXs, which arenonselenium containing enzymes in which cysteine replaces selenocysteineat their active site (Ursini et al., 1995). However, with the exceptionof phospholipid hydroperoxide GPX and perhaps pl•GPX, the enzymesexhibit a strong preference for glutathione as a source of reducingequivalents.

Despite the presence of SOD, CAT, and GPX, the body's antioxidantdefense system is constantly subject to oxidative stress, and itsability to produce SOD, CAT, and GPX is compromised by the aging processand can further be impaired by inflammation, microbial or viralinfections, the progression of cancer and neurological disorders, andother pathological conditions that produce, are caused by, or areexacerbated by oxidative stress.

Herb-Containing Compositions of the Invention

One object of the present invention is to provide new and usefulcompositions that alleviate oxidative stress in a subject. Anotherobject of the present invention is to provide compositions that assistthe body's natural antioxidant defense system(s) to alleviate the harmassociated with oxidative stress. A further object of the presentinvention is to provide new compositions that upregulates the levels ofSOD, CAT, and GPX, lowers the concentrations of free radicals and ROS,and decreases the rate of lipid peroxidation. Also, an object of thepresent invention is to provide dietary supplement compositions thatalleviate oxidative stress as well as provide the body with otherhealthful benefits. Yet another object of the present invention is toformulate a composition in the form of a tonic or capsule, with thebeneficial effects of alleviating oxidative stress that is of asufficient concentration that may be easily implemented as part of adaily supplement regime. As noted above, the compositions of theinvention provide for an overall net decrease in oxidative stressexperienced by the mammal, with minimal undesirable side-effects.Advantageously, the composition itself provides fewer side effects thanmay be associated with each of the individual active agents in thecomposition.

These and other objects of the present invention will become morereadily appreciated and understood from a consideration of the followingdetailed description of the present invention.

The present invention provides compositions derived from plant sourcesthat induce the body's production of the antioxidants SOD, CAT, and GPX,and to a method of alleviating oxidative stress through use of thesecompositions. The compositions provide for efficient upregulation ofthese antioxidant enzymes enzymes, but advantageously the compositionhas lowered side-effects, i.e., reduced toxicity in the combinationrelative to the toxicity of each active agent when administered alone.

Observations related to lipid peroxidation are a good measure as to theamount of oxidative stress in a subject. Ideally, natural dietarysupplements that induce production of SOD, CAT and GPX should also havethe effect of reducing the concentration of free radicals and ROS in thebody and result in decreased rates of lipid peroxidation. However, ithas been observed that some formulations of dietary supplements thatinduce antioxidant production may contemporaneously cause an increase inthe concentration of ROS leading to increased rates of lipidperoxidation. In other words, while some supplements may achieve anincrease in the body's production of SOD, CAT, and GPX, it leaves thebody with an overall net increase in oxidative stress. The presentcompositions do not add to the oxidative stress of the body.

Accordingly, choosing an active agent based simply on its ability tocause the induction of antioxidant production, per se, may not be anadequate selection criterion to achieve the overall desired goal ofalleviating oxidative stress. Furthermore, plant extracts, which aretypically the ingredients of natural dietary supplements, generally havetheir respective undesirable side-effects, that also need to be properlybalanced in an overall formulation that accomplishes a dilution ofindividual undesirable effects. For example, research has indicated thatan ethanol extract of Bacopa monniera produces toxicity in the brineshrimp lethality assay at about 300 mg/L (D'Souza et al. PhytotherapyRes. 2002, vol 16, 197-8) and enhances thyroid function, which may be anundesirable side effect (Kar et al. J. Ethnopharmacol. 2002, vol 81,281-5).

The foregoing problems, among others, have been resolved by the presentinvention. Specifically, the present invention provides compositionscontaining appropriate mixtures of plant extracts are to safely induceantioxidant production to achieve an overall net decrease in oxidativestress, while at the same time diluting or reducing the undesirableside-effect and toxicity profiles associated with the individualextracts that make up the composition or any pharmaceutically acceptableformulation thereof.

The mixture contemplated by the present invention is comprised of thefollowing nine plant derived active agents, which are listed in thefollowing table along with the desired amounts of each agent. In oneaspect, an antioxidant inducing agent further comprises any of thefollowing nine plant derived active agents, which are incorporated intothe antioxidant-inducing preparation in currently preferredconcentrations (wt/wt) ranging from about 0.001 mg to about 1000 g,preferably about 0.1 mg to about 10 g, more preferably about 1 mg toabout 2 grams, and most preferably about 50 mg to about 500 mg.

TABLE 1 AMOUNT Per dosage ACTIVE AGENTS unit POSSIBLE UNDESIRED EFFECTSBacopa monniera extract 0.001 mg Considered very safe, but showscytotoxicity to 1000 g in cultured cells (100 mg/kg) and anticanceractivity (D'Souza et al., Phytotherapy Res. 2002, vol 16, 197-8);enhances thyroid function (200 mg/kg) (Kar et al. J. Ethnopharmacol.2002, vol 81, 281-5) Gotu Kola powder 0.001 mg Sedative andantidepressive effects; Possible to 1000 g hypotensive effect (PDR forHerbal Medicines (First Edn). Medical Economics Co., 1998, 729-30)Ashwagandha powder 0.001 mg Considered very safe, but shows to 1000 gtranquilizing and hypotensive effects at 25 mg/kg (Mishra et al. Altern.Med. Rev. 2000, vol 5, 334-346); (Malhotra et al., Indian J. PhysiolPharmacol. 1965, vol 9, 127-136) Green tea 98% 0.001 mg Stimulant(caffeine); possible gastric irritant Polyphenols 45% EGCG to 1000 g(PDR for Herbal Medicines (First Edn). Medical Economics Co., 1998,710-1) Turmeric extract 95% 0.001 mg Possible gastric irritant; possibleantifertility to 1000 g effects at 125 mg/kg (PDR for Herbal Medicines(First Edn). Medical Economics Co., 1998, 786-7) Milk Thistle extract0.001 mg Considered very safe, but causes increases 70-80% to 1000 gcell division in vitro (PDR for Herbal Medicines (First Edn). MedicalEconomics Co., 1998, 1138-9); May be proinflammatory at high doses of250 mg/kg (Johnson et al. Planta Med. 2003, vol. 69, pp. 44-49) Aloevera powder 0.001 mg Laxative effect; Gastrointestinal cramping to 1000g (PDR for Herbal Medicines (First Edn). Medical Economics Co., 1998,630-3); inhibits thyroid function at 125 mg/kg (Kar et al., J.Ethnopharmacol. 2002, vol 81, 281-5) Ginko biloba leaf extract 0.001 mgMild gastrointestinal irritant; hypersensitivity to 1000 g reactions;may interfere with antithrombotic therapy at 240 mg/day (PDR for HerbalMedicines (First Edn). Medical Economics Co., 1998, 871-3) N-AcetylCysteine 0.001 mg Considered Safe to 1000 g

TABLE 2 ACTIVE AGENTS DOSAGE/DAY Bacopa monniera extract 10-4,000 mgMilk Thistle extract 70-80% 15-6,000 mg Ashwagandha powder 10-4,000 mgTurmeric extract 95% 5-2,000 mg Gotu Kola powder 10-4,000 mg Aloe verapowder 10-4,000 mg Green tea, 98% Polyphenols 45% EGCG 5-2,000 mg Ginkobiloba leaf extract 5-2,000 mg N-Acetyl Cysteine 50-5,000 mg

As shown in Table 1, many of the plant extracted active agents listedhave the possibility of undesirable side-effects at high doses. However,by using a combination of some or all of these nine active agents in therespective amounts as listed in Table 2 to formulate a plant basedcomposition (e.g., an oral dietary supplement), the desirable effectsare additive and in certain embodiments, even synergistic. The overalleffect then, is to upregulate at least one antioxidant enzyme, e.g.,SOD, CAT, and GPX, while decreasing the concentration of free radicalsand ROS and the rate of lipid peroxidation and other undesirablechemical reactions that result in oxidative stress on the body. Forexample, tissue level of thiobarbituric acid reactive chemical species(e.g., plasma TBARS) are reduced after administration of a compositionof the invention to a subject. Furthermore, the likelihood of observingthe undesired side-effects listed for each respective active agent ismore remote as each extract is diluted out by a factor of up to nine(9).

Further, it is expected that the above-described active agents, in theamounts listed, will provide a combined remedy for oxidative stress thatmay be processed, in one embodiment, into a dosage unit for oraladministration. This dosage unit is then administered, as a tablet,capsule, gel cap, pellet (globule), or in other carrier suitable fororal administration. Alternatively, the composition could be madeavailable as a powder to be mixed with a suitable liquid, such as water,to form a tonic. In one embodiment, the formulation of theherb-containing composition of the invention is an oral dietarysupplement. As such, an efficient, proper, and effective balance ofthese active agents can be formulated as to provide a composition thatcan be administered as a suitable daily oral dietary supplement.

In one embodiment, the herb-containing composition of the inventioncontains at least two (2) of the components (e.g., ingredients)summarized in Table 2. In some embodiments, the herb-containingcomposition of the invention contains two (2), three (3), four (4), five(5), six (6), seven (7), eight (8), or nine (9) of the componentssummarized in Table 2. The herb-containing composition of the inventioncan contain the components summarized in Table 2 in any quantity, orcombination, suitable to give the desired oxidant preventative,therapeutic, or alleviating effect.

Antioxidant Properties and Uses of the Compositions of the Invention

The invention provides a method of preventing, alleviating or treatingoxidative stress in a subject. The active agents of the invention areformulated into a composition that retains the prophylactic andtherapeutic antioxidant inducing properties of the individual activeagents, providing an additive or even synergistic antioxidant inducingeffect relative to the effect of each active alone, while alsodecreasing the toxic side effect(s) to a subject, of the individualactive agents of the compositions. The compositions of the invention areuseful to eradicate free and bound radical reactions presently takingplace or it may be used as prophylaxis against pathological free orbound radical reactions, which may occur as a result of a possibleoxidant promoting incident (e.g., ischemic injury).

The present invention provides compositions for increasing the levels ofantioxidants, via alteration of the activity level of SOD, CAT, and GPXenzymes in the body. The composition provides in one embodiment, amixture of herbal extracts of Bacopa monniera (B. monniera or Bacopa),which contains a high percentage of the active chemicals bacosides A &B. Ingestion of Bacopa induces SOD, CAT, and GPX and provides thebeneficial activities thereof, with pronounced results in the brain.Studies have indicated that the bacosides also increase protein andserotonin levels, while decreasing norepinephrine concentration in thehippocampus, hypothalamus, and cerebral cortex. Bacopa thus reduces theneurodegeneration in the brain that is caused by oxidative stressrelated to the accumulation of neurotoxic free radicals in the brain.Accordingly, it may be used to alleviate symptoms of neurodegenerativedisorders, such as memory loss, Alzheimer's disease, and Parkinson'sdisease, and even aging.

Bacopa monniera

Bacopa monniera (common names: water hyssop and Brahmi) is a creepingperennial that thrives in warmer temperate climates. The genus Bacopaincludes over 100 species of aquatic herbs distributed throughout thewarmer regions of the world. The plant is a profusely branched herb,rooting at the nodes and forming dense mats. B. monniera extract(Bacopin®) is a standardized extract prepared from the leaves of the B.monniera plant (Sabinsa Corporation, Piscataway, N.J., USA). It isstandardized for a minimum of 20% bacosides A & B, the activeingredients beneficial in the support of cognitive functions. Otherextracts of the B. monniera plant standardized for a greater minimumlevels of bacosides A & B (e.g., 30%, 40%, 50%, etc.) are useful in thecompositions of the present invention and can be prepared by extractiontechniques known in the art. The pharmacological effects of B. monnierapreparations/extracts also include antioxidant, anti-inflammatory,cardiotonic and anticancer effects. Extract of B. monniera iscommercially available, e.g., Viable Herbal Solutions (Morrisville, Pa.,USA).

In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 10 mg to about 4,000 mg B.monniera extract is administered to a subject daily using anherb-containing composition of the invention (See generally, Table 2).In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 50 mg to about 3,000 mg B.monniera extract is administered to a subject daily using anherb-containing composition of the invention. In one embodiment of themethod preventing, alleviating or treating oxidative stress in asubject, from about 100 mg to about 2,000 mg B. monniera extract isadministered to a subject daily using an herb-containing composition ofthe invention. In one embodiment of the method preventing, alleviatingor treating oxidative stress in a subject, at least about 200 mg B.monniera extract is administered to a subject daily using anherb-containing composition of the invention. In one embodiment, the B.monniera extract of the herb-containing composition is Bacopin®. In oneembodiment, the herb-containing composition of the invention contains aB. monniera extract standardized for a at least about 20% bacosides A &B. In another embodiment, the herb-containing composition of theinvention contains a B. monniera extract standardized for at least about30% bacosides A & B. In another embodiment, the herb-containingcomposition of the invention contains a B. monniera extract standardizedfor at least about 40% bacosides A & B. In another embodiment, theherb-containing composition of the invention contains a B. monnieraextract standardized for at least about 50% bacosides A & B.

Milk Thistle

Milk thistle (botanical name; Silybum marianum; other common names:Marian, Silybum, Silymarin) is a fine, tall plant, about the size of theCotton Thistle, with cut into root-leaves, waved and spiny at themargin, of a deep, glossy green, with milk white veins, and is found notuncommonly in hedgebanks and on waste ground. Useful parts of the plantinclude, e.g., the whole herb, root, leaves, seeds and hull. Milkthistle seeds contain a bioflavonoid complex known as silymarin.Silymarin is an extract of the seeds of the milk thistle plant. Astandardized extract should be 80% silymarin (the active ingredient).This constituent is responsible for the medical benefits of the plant.Silymarin is made up of three parts: silibinin, silidianin, andsilicristin. Silibinin is the most active and is largely responsible forthe benefits attributed to silymarin. As with other bioflavonoids,silymarin is a powerful antioxidant. Milk thistle extract is useful toprotect or reverse damage to liver cells from toxins (e.g., alcohol,drugs, pesticides, poisons), to promote the regeneration of liver cells,to prevent or treat liver disease (e.g., liver cirrhosis, chronichepatitis, and diabetes due to cirrhosis), indigestion, and cancer.Silymarin's effect in preventing liver destruction and enhancing liverfunction relates largely to its ability to inhibit the factors that areresponsible for hepatic damage, i.e., free radicals and leukotrienes,coupled with an ability to stimulate liver protein synthesis. Milkthistle (80% silymarin) extract is commercially available, e.g.,Stayleaner.com (Las Vegas, Nev., USA).

In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 15 mg to about 6,000 mg milkthistle extract (70%-80%) is administered to a subject daily using anherb-containing composition of the invention (See generally, Table 2).In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 50 mg to about 5,000 mg milkthistle extract (70%-80%) is administered to a subject daily using anherb-containing composition of the invention. In one embodiment of themethod preventing, alleviating or treating oxidative stress in asubject, from about 100 mg to about 3,000 mg milk thistle extract(70%-80%) is administered to a subject daily using an herb-containingcomposition of the invention. In one embodiment of the methodpreventing, alleviating or treating oxidative stress in a subject, atleast about 300 mg milk thistle extract (70%-80%) is administered to asubject daily using an herb-containing composition of the invention.

Ashwagandha

Ashwagandha (botanical names: Withania somnifera and Physalis flexuosa;other common names: winter cherry, Ashgandh, Achuvagandi,Amikkira-gadday, Amkulang-kalang, Amukkira-kilzhangu, Amukran-kizhangu,Asagandha, Asana, Asgandh, Asundha, Asvagandhi, Fatarfoda,Hirimaddina-gadday, Hirre-gadday, Penneroo-gadda, Pevette, Sogade-beru,Indian ginseng) is an erect branched shrub native to India, Pakistan andSri Lanka. Ashwagandha preparations are useful to promote relaxation,bone marrow and women's health (e.g. stabilizes fetus and regenerateshormones), to enhance mental function (e.g., memory and concentration),as an aphrodisiac, and to treat fatigue, stress, cough, infertility,tissue inflammation, cancer, infectious disease, anxiety disorders,panic attacks, rheumatism, arthritis, pain, manic depression, alcoholicparanoia, and schizophrenia, fever, insomnia, infertility, aging, skininflammations and disorders, alcoholism, Alzheimer's disease, anemia,carbuncles, convalescence, emaciation, HIV support, AIDS, immune systemproblems, lumbago, multiple sclerosis, muscle energy loss, paralysis,skin afflictions, swollen glands, ulcers, as well as breathingdifficulties and as a diuretic. Ashwaganda powder is commerticallyavailable, e.g., iHerb Inc. (Monrovia, Calif., USA).

In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 10 mg to about 4,000 mgAshwagandha powder is administered to a subject daily using anherb-containing composition of the invention (See generally, Table 2).In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 50 mg to about 3,000 mgAshwagandha powder is administered to a subject daily using anherb-containing composition of the invention. In one embodiment of themethod preventing, alleviating or treating oxidative stress in asubject, from about 100 mg to about 2,000 mg Ashwagandha powder isadministered to a subject daily using an herb-containing composition ofthe invention. In one embodiment of the method preventing, alleviatingor treating oxidative stress in a subject, at least about 200 mgAshwagandha powder is administered to a subject daily using anherb-containing composition of the invention.

Turmeric

Turmeric extract 95% is prepared from the root or rhizome of the Curcumalonga plant (common names: Curcuma, Turmeric, Ukon, Goeratji, Kakoenji,Koenjet, Kondin, Kunir, Kunyit, Oendre, Rame, Renet, Temu kuning, Temukunyit, Tius. Curcumin). C. longa is a perennial plant native to India.A compound called curcumin is a potent extract of the root, and has beenattributed a wide range of therapeutic benefits. Turmeric extract isuseful as an antioxidant, anti-inflammatory, anti-mutagenic agent,anti-cancer agent, cholagogueue, depurative, diuretic, fumitory,hemostatic agent, hepatoprotective agent, lactagogue, stomachic, tonic,and vulnerary. Turmeric preparations are useful to protect the liverfrom toxins, to reduce platelet aggregation, to prevent or treatinflammatory disease, inflammation, arthritis, psoriasis, cancer (e.g.,prostate cancer and breast cancer), pain, Alzheimer's Disease,cardiovascular disease (e.g., arteriosclerosis and atherosclerosis).Turmeric extract that is standardized to 95% curcumin contains turmeric(with 95% curcumin). Turmeric extract 95% is commercially available,e.g., EZ-FITNESS (Northborough, Mass., USA).

In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 5 mg to about 2,000 mgTurmeric extract (95%) is administered to a subject daily using anherb-containing composition of the invention (See generally, Table 2).In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 10 mg to about 1,000 mgTurmeric extract (95%) is administered to a subject daily using anherb-containing composition of the invention. In one embodiment of themethod preventing, alleviating or treating oxidative stress in asubject, from about 50 mg to about 500 mg Turmeric extract (95%) isadministered to a subject daily using an herb-containing composition ofthe invention. In one embodiment of the method preventing, alleviatingor treating oxidative stress in a subject, at least about 100 mgTurmeric extract (95%) is administered to a subject daily using anherb-containing composition of the invention.

Gotu kola

Gotu kola (botanical names: Hydrocotyle asiatica, Centella asiatica;other common names: Centella, March Pennywort, Indian Pennywort,Hydrocotyle, Brahmi (Sanskrit), Luei Gong Gen (Chinese)) is a slender,creeping perennial plant that grows commonly in swampy areas of India,Sri Lanka, Madagascar, South Africa and the tropics. Gotu kola isdistinct from the kola nut. Gotu kola powder is prepared from the leavesand aerial parts of the plant and used for medicinal purposes. Gotu kolapowder is useful to promote relaxation, to enhance mental function(e.g., memory and concentration), to promote tissue healing (e.g.,wounds, skin, other connective tissues, lymph tissue, blood vessels, andmucous membranes) and to treat fatigue, anxiety, attention deficitdisorder, insomnia, skin inflammations, leprosy, cancer, arthritis(e.g., psoriatic arthritis, anklylosing spondvlitis, and rheumatoidarthritis), hemorrhoids, tuberculosis, high blood pressure, congestiveheart failure, venous insufficiency (pooling of blood in the veins,usually in the legs), sore throat, hepatitis, syphilis, stomach ulcers,epilepsy, diarrhea, fever, and asthma and as a mild diuretic. Gotu kolapowder is commercially available, e.g., @Internatural (Twin Lakes, Wis.,USA).

In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 10 mg to about 4,000 mg Gotukola powder is administered to a subject daily using an herb-containingcomposition of the invention (See generally, Table 2). In one embodimentof the method preventing, alleviating or treating oxidative stress in asubject, from about 25 mg to about 2,000 mg Gotu kola powder isadministered to a subject daily using an herb-containing composition ofthe invention. In one embodiment of the method preventing, alleviatingor treating oxidative stress in a subject, from about 50 mg to about1,000 mg Gotu kola powder is administered to a subject daily using anherb-containing composition of the invention. In one embodiment of themethod preventing, alleviating or treating oxidative stress in asubject, at least about 200 mg Gotu kola powder is administered to asubject daily using an herb-containing composition of the invention.

Aloe vera

Aloe vera (common names: medicinal aloe, burn plant, Barbados aloe,unguentine cactus) is a perennial plant; the strong, fibrous rootproduces a rosette of fleshy basal leaves as in the agave butconsiderably smaller that grows wild in East and South Africa and alsocultivated in the West Indies and other tropical areas. Aloe containsanthraquinone glycosides, resins, polysaccharides, sterols, gelonins,and chromones, which contribute to the herbs medicinal properties. Aloepreparations, e.g., sap or powder, are useful as emollients, purgatives,a vulnerary agent, tonic, demulcent, vermifuge, antifungal, emmenagogue.Aloe preparations are useful to treat burns, piles, sunburn, wrinkles,headache, insect bites, skin irritations, cuts, ulcers, sores, herpes,jaundice, bursitis, canker sores, sore gums, poison ivy, inflammation,gastritis, and cancer. Aloe vera powder is commercially available, e.g.,Red Lion International Trading & Brokerage Co. (Fullerton, Calif., USA).

In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 10 mg to about 4,000 mg Aloevera powder is administered to a subject daily using an herb-containingcomposition of the invention (See generally, Table 2). In one embodimentof the method preventing, alleviating or treating oxidative stress in asubject, from about 25 mg to about 2,000 mg Aloe vera powder isadministered to a subject daily using an herb-containing composition ofthe invention. In one embodiment of the method preventing, alleviatingor treating oxidative stress in a subject, from about 50 mg to about1,000 mg Aloe vera powder is administered to a subject daily using anherb-containing composition of the invention. In one embodiment of themethod preventing, alleviating or treating oxidative stress in asubject, at least about 200 mg Aloe vera powder is administered to asubject daily using an herb-containing composition of the invention.

Green Tea

Green tea extracts are useful in the compositions of the presentinvention. In some embodiments of the compositions of the invention, theGreen tea extract is standardized for polyphenols. For example, Greentea, 98% polyphenols containing 45% polyphenols such as polyphenol(−)-epigallocatechin gallate (EGCG) is prepared from the leaf of the teaherb Camellia sinensis. Polyphenols, e.g., EGCG, in green tea are usefulto protective against certain cancers, and they are also potentantioxidants. Green tea preparations are useful to promote immunefunction and to prevent and treat high cholesterol, heart disease,infection (e.g., Staphylococcus aureus infection, skin infection,bacterial infection, viral infection), acne, aging, immune disorders,dental caries, periodontitis, halitosis, dandruff, cancer,cardiovascular disease (e.g., hypertension, thrombosis,arteriosclerosis), diabetes, elevated blood glucose, diseases of thealimentary canal and respiratory system, influenza hepatitis, liverdisease. Green tea extracts are commercially available, e.g., HunanKinglong Bio-Resource Co., Ltd., (Xingsha, Changsha, Hunan, P. R.China).

In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 5 mg to about 2,000 mg Greentea (98% polyphenols, 45% EGCG) is administered to a subject daily usingan herb-containing composition of the invention (See generally, Table2). In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 10 mg to about 1,000 mg Greentea (98% polyphenols, 45% EGCG) is administered to a subject daily usingan herb-containing composition of the invention. In one embodiment ofthe method preventing, alleviating or treating oxidative stress in asubject, from about 50 mg to about 500 mg Green tea (98% polyphenols,45% EGCG) is administered to a subject daily using an herb-containingcomposition of the invention. In one embodiment of the methodpreventing, alleviating or treating oxidative stress in a subject, atleast about 100 mg Green tea (98% polyphenols, 45% EGCG) is administeredto a subject daily using an herb-containing composition of theinvention.

Ginkgo biloba

Ginkgo biloba (common name: Maidenhair tree) is a dioecious tree. Ginkgoleaf extracts have been shown to have a wide range of biologicalactivities. The leaf extract utilized in medicine is standardized in amulti-step procedure designed to concentrate the desired activeprinciples from the plant. These extracts contain approximately flavoneglycosides (primarily composed of quercetin, kaempferol, andisorhamnetin) and terpene lactones (ginkgolides A, B, and C, andbilobalide). Other constituents typically include proanthocyanadins,glucose, rhamnose, organic acids, D-glucaric acid and ginkgolic acid (atmost 5 ppm ginkgolic acids). The complex extract itself, rather than asingle isolated component, is believed to be responsible for Ginkgo'sbiological activity. The flavonoid complex can remove free radicals inthe peripheral and/or cerebral vascular systems, and inhibit lipidperoxidation. Ginko biloba extract is commercially available, e.g.,iHerb Inc. (Monrovia, Calif., USA).

Some of the many valuable effects ginkgo has are stabilizing cellmembranes, reducing free radical damage, improving blood circulation andenhancing oxygen and glucose use. Ginkgo is very beneficial for thebrain, nerves and blood vessels. It is useful to improve short-termmemory Ginkgo biloba has been used to improve cerebral blood circulationand to protect the nerves against damaging free radicals. Ginkgo leafhelps to maintain integrity and permeability of cell walls by inhibitinglipid peroxidation of membranes. Ginkgo extracts are useful to preventor treat age-related physical and mental deterioration (e.g.,Alzheimer's Disease and age-related dementia), cardiovascular disease,cerebral vascular insufficiency and impaired cerebral performance,congestive symptoms of premenstrual syndrome, allergies, age-relatedvision loss, depression, Raynaud's disease, peripheral vascular disease,intermittent claudication, vertigo, equilibrium disorder, prevention ofaltitude sickness, tinnitus (ringing in the ear), liver fibrosis,macular degeneration, asthma, graft rejection, and immune disorders thatinduce toxic shock.

In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 5 mg to about 2,000 mg G.biloba leaf extract is administered to a subject daily using anherb-containing composition of the invention (See generally, Table 2).In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 10 mg to about 1,000 mg G.biloba leaf extract is administered to a subject daily using anherb-containing composition of the invention. In one embodiment of themethod preventing, alleviating or treating oxidative stress in asubject, from about 50 mg to about 500 mg G. biloba leaf extract isadministered to a subject daily using an herb-containing composition ofthe invention. In one embodiment of the method preventing, alleviatingor treating oxidative stress in a subject, at least about 200 mg G.biloba leaf extract is administered to a subject daily using anherb-containing composition of the invention.

N-Acetyl Cysteine

N-Acetyl Cysteine (NAC) is an acetylated form of the amino acidcysteine. NAC is an antioxidant, an antitoxin and immune supportsubstance, and is found naturally in foods. There have been severalinstances wherein NAC has been demonstrated to be an antioxidant. NAC iscommercially available, e.g., Doctor's Trust Vitamins (Orlando, Fla.,USA).

NAC reacts very slowly with superoxide or the hydrogen peroxide freeradicals. It can be seen from the rate constants that GSH is a moreeffective antioxidant against the hydroxyl radical in comparison to NAC(GSH K2=8.8×109, NAC K2=1.36×1010 at a pH of 1.0). NAC will inhibit HOClat physiological concentration in a 3:1 ratio (respectively).

NAC is a precursor for glutathione, an important antioxidant thatprotects cells against oxidative stress. In addition to maintainingintracellular glutathione levels, NAC supplementation has been shown tosuppresses Human Immunodeficiency virus (HIV) replication, to beprotective against cell damage caused by chemotherapy and radiationtherapy, to be immune enhancing, to protect against toxins asacetametaphen and other drugs, mercury, lead, and others, and ismucolytic, that is, it breaks up mucus seen in bronchoulmonary diseaseas cystic fibrosis, chronic bronchitis, asthma, gastritis, heart attack,angina pectoris, chronic obstructive pulmonary disease, prevention ofkidney damage during coronary angiography, Unverricht-Lundborg disease,pseudoporphyria, and pneumonia. It has also been shown to offerprotection against the superoxide free radical in porcine aorticendothelial cells and protects animals against paracetamolhepatotoxicity.

In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 50 mg to about 5,000 mgN-acetyl cysteine is administered to a subject daily using anherb-containing composition of the invention (See generally, Table 2).In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, from about 100 mg to about 4,000 mgN-acetyl cysteine is administered to a subject daily using anherb-containing composition of the invention. In one embodiment of themethod preventing, alleviating or treating oxidative stress in asubject, from about 250 mg to about 2,000 mg N-acetyl cysteine isadministered to a subject daily using an herb-containing composition ofthe invention. In one embodiment of the method preventing, alleviatingor treating oxidative stress in a subject, at least about 500 mgN-acetyl cysteine is administered to a subject daily using anherb-containing composition of the invention.

Free radical damage on the clinical level will be determined by thelocation of the preponderance of the oxidants generated, the type ofoxidants, and the generation of pathological prostaglandins. Theadministration of compositions of the present invention is dependentupon where pathological free radical reactions are taking place. In oneembodiment, the compositions of the present invention are orallyadministered to a subject in need thereof. Measuring the rate of theappearance of oxidation products can assess the effectiveness of thecompositions of the invention. Effectiveness can also be monitored inpatients by their clinical progress.

In one aspect, the invention provides a method of increasing theantioxidant activity level of a mammalian subject in need thereof, byincreasing the level of enzyme activity of at least one enzyme, e.g.,superoxide dismutase; catalase; and glutathione peroxidase, byadministering to the subject an effective amount of anantioxidant-promoting composition of the invention, wherein theincreased enzyme activity decreases the tissue damage caused bypathological free radicals. In one embodiment, the tissue damage causedby pathological free radicals occurs in a mammalian subject with adisease or condition selected from the group which includes, e.g.,inflammation; infection; atherosclerosis; hypertension; cancer;radiation injury; neurological disease; neurodegenerative disease;ischemia/reperfusion injury; aging; wound healing; glutathionedeficiency; acquired immunodeficiency syndrome; sickle cell anemia; anddiabetes mellitus. In one embodiment of the method, theantioxidant-promoting composition is administered as an oral dietarysupplement.

In another embodiment the invention provides a method of reducing theplasma level of c-reactive protein in a mammalian subject, byadministering to the subject an effective amount of anantioxidant-promoting composition of the invention. In one embodiment ofthe method, the antioxidant-promoting composition is administered as anoral dietary supplement. In one embodiment of the method, the plasmalevel of c-reactive protein decreases by at least 10% or more in a 30day period. In one embodiment of the method, the plasma level ofc-reactive protein decreases by at least 20% or more in a 30 day period.

In another embodiment, the invention provides a method of reducing theblood pressure in a mammalian subject, by administering to the subjectan effective amount of an antioxidant-promoting composition of theinvention. In one embodiment of the method, the antioxidant-promotingcomposition is administered as an oral dietary supplement.

ROS and Human Health

Our bodies are continuously exposed to free radicals and other ROS, fromboth external sources (sunlight, other forms of radiation, pollution)and generated endogenously; oxidative stress and ROS-mediated tissueinjury is a final common pathway for a number of pathological processes.Oxidative stress results in increased immune system activity, whichleads to inflammation, recruitment of more immune cells, and release ofcytokines and acute phase proteins that further exacerbate the stress onthe body.

Cytokines

It is postulated that in conditions where there is excessive freeradical production or infection (e.g. AIDS), there is a severealteration of interleukin-2 (IL-2) production, which secondarily occursdue to glutathione (GSH) depletion. IL-2 is a glycoprotein, which isproduced in response to mitogens and antigenic stimuli; it and othercytokines show a multiplicity of functions. Glutathione levels regulatethe alpha chain, the larger of the two IL-2 receptors. Decreasing GSHlevels would decrease the affinity of IL-2 to its correspondingreceptors; consequently there would be a compromise in the function ofIL-2. It is postulated that maintenance of GSH levels by the use of thecompositions of the present invention would allow IL-2 and its receptorsto elicit the normal immunological response for this particularinterleukin.

Excessive oxidative stress results in amplified production of TNF-alphaand IL-6. IL-6 initiates and encourages the production of acute phaseproteins such as c reactive protein, serum amyloid A protein,fibrinogen, and mannan-binding lectin. IL-1, IL-6, and TNF-alphastimulate, for example, CRP synthesis by inducing hepatic geneexpression, which triggers a variety of inflammatory responses andassociated pathologies (see, Albert M A. The role of C-reactive proteinin cardiovascular disease risk. Curr Cardiol Rep 2000; 2(4):274-9). CRPis also a mediator of the complement system, part of the innate immuneresponse (see, Yuan G, Expression of C5aR (CD88) of synoviocytesisolated from patients with rheumatoid arthritis and osteoarthritis.Chin Med J (Engl). 2003 September; 116(9):1408-12). The complementsystem provides further stimulus of TH1 and TH2 adaptive immuneresponses, which adds to the inflammatory response.

Production of tumor necrosis factor-alpha (TNF-alpha) by macrophages isstimulated by free radicals or oxidants (Chaudhri, G. and Clark, I. A.:J Immunol, vol. 143, 1990-1294, No. 4, 1989). TNF-alpha induces oxidantproduction by stimulating leukocytes, releasing arachidonic acid fromleukocytes and releasing lysosomes. Therefore, enhancing plasma levelsof antioxidants by administering the compounds of the present inventionwould decrease the production of TNF-alpha. It follows that, themaintenance/increase in antioxidant potential by administering thecompositions of the present invention to a subject as described canprevent or treat cytokine-mediated tissue injury.

Inflammation

Inflammation can arise from infective agents (e.g. virion), trauma,chemical agents, immune reactions, metallic agents, and ionizing orthermal agents. The sine qua non of inflammation is heat, redness,edema, pain and loss of function (e.g., of the surrounding tissue). Inany type of inflammation, characteristic inflammatory cells can befound, for example leukocytes, eosinophils, and macrophages/macrocytes.Each of these cell types produce radicals as part of a programmedresponse. Also as part of that “programmed” response are the productionof inflammatory cytokines, such as TNF-alpha, CM-CSF and IL-2 and IL-6.These particular cytokines promote the production of oxidants such asnitric oxide and other reactive compounds. Oxidants are also generatedas a byproduct of prostaglandin (PG) production, which is part of thepropagation and amplification of the inflammatory process. Platelets arealso involved in the inflammatory process by virtue of their ability toact as a plug (as in a clot); but also due to their liberation ofplatelet activating factor (PAF). PAF liberates arachidonic acid fromleukocytes.

The production of prostaglandins is dependent upon the free radical tone(or concentration) of the microenvironment and metabolite synthesis. Bydecreasing the free radical tone and PG free radical intermediatemetabolites, it is postulated that the pathological production ofprostaglandins would be reduced, the amplification effect that PGs haveas a role in the inflammatory process could be limited. Theoretically,either the lipooxygenase limb or the cyclooxygenase limb of theprostaglandin pathway could be affected by increased cellularantioxidants.

Free radicals or oxidants also have a plethora of different effects onthe tissue in which it occurs, e.g., membrane damage, platelet adhesion,blood vessel intimal damage, etc. By increasing the antioxidant levelsin areas where inflammation is occurring, it is postulated that thepropagatory effect, tissue damage and pathologic physiologic reactionswould be curtailed as well. The NF-KB transcription protein regulatesthe expression of a number of genes for proteins and cytokines involvedin the inflammatory process (Baeuerle, P. A. and Baltimore, D.: Science,vol. 242, October, 1988). The activity and affinity that the NF-KBprotein has for DNA is also regulated by GSH level (Staal, F. J. T, etal.: Proc. Natl. Acad. Sci., USA, vol. 87, pp 9943-9947, December 1990;Duh, E. J., et al.: Proc. Natl. Sci. Acad., USA, vol. 86, p 5974-5978,1989). Enhancing levels of GSH decreases the activity and binding ofNF-KB to DNA. By enhancing GSH levels, those cytokines and proteinsinvolved in the inflammatory process would be decreased. Themaintenance/increase of antioxidant potential by administering thecompositions of the invention to a subject can, therefore, prevent ortreat inflammation-mediated injury.

One important marker for inflammation is c reactive protein (CRP), amajor acute phase response protein synthesized in the liver in responseto the elaboration of acute phase response cytokines, such asinterleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factoralpha (TNF-alpha). Other associated acute phase proteins include serumamyloid A protein, fibrinogen, and mannan-binding lectin.

CRP concentrations are elevated in almost all inflammatory, infectious,and neoplastic diseases. Specific conditions include rheumatologicdiseases (e.g., systemic lupus erythematosus, Sjögren's syndrome,rheumatoid arthritis), vasculitides (e.g., Wegener's granulomatosis),and chronic infections (e.g., tuberculosis, endocarditis). Certainmalignant neoplasms, such as solid tumors, also may be associated withan elevated CRP level. Because CRP has a long half-life, CRP levelscorrelate well to its synthesis induced by persistent inflammation.Elevated levels of CRP are also associated with cardiovascular diseasessuch as endocarditis, angina pectoris, and myocardial infarction (see,http://www.americanheart.org/presenterjhtml?identifier=4648 andhttp://www.americanheart.org/presenterjhtml?identifier=3006541). Certainpathogens have been linked to atherogenesis and the development ofclinically relevant coronary atherosclerosis. For example,cytomegalovirus (CMV), Herpes simplex virus (HSV) Chlamydia pneumoniae,and Helicobacter pylori have been associated with coronary arterydisease (CAD), through inducement of vascular inflammation in additionto other mechanisms. This finding suggests that CMV contributes toatherogenesis by provoking an inflammatory response. CRP levelscorrelate with the clinical severity of CAD and with coronary events inboth the acute and subacute phases of myocardial ischemia. Patients whoare hospitalized for the treatment of unstable angina and have CRPconcentrations above 0.3 mg/dL have significantly more ischemic episodesin the hospital than patients with lower CRP levels (see, Morrow D,Rifai N, Antman E M, et al. C-reactive protein is a potent predictor ofmortality independently of and in combination with troponin T in acutecoronary syndromes: a TIMI 11A substudy. Thrombolysis in MyocardialInfarction. J Am Coll Cardiol 1998; 31(7):1460-5). CRP concentrationsare significantly lower in patients with stable angina pectoris than inthose with unstable angina pectoris or an acute coronary syndrome.Patients with chronic stable angina who have stable, low CRP levels overtime have fewer subsequent cardiovascular events during follow-up (see,Bogaty P, Poirier P, Simard S, et al. Biological profiles in subjectswith recurrent acute coronary events compared with subjects withlong-standing stable angina. Circulation 2001; 103(25):3062-8). On theother hand, in patients with unstable angina pectoris, elevated CRPlevels are strong predictors of plaque instability.

Many trials have confirmed the association between high levels of CRPand the risk of future coronary events such as MI and sudden cardiacdeath. In the European Concerted Action on Thrombosis and Disabilitiesstudy (Bolibar I, von Eckardstein A, Assmann G, et al. Short-termprognostic value of lipid measurements in patients with angina pectoris.The ECAT Angina Pectoris Study Group: European Concerted Action onThrombosis and Disabilities. Thromb Haemost 2000; 84(6):955-60),elevation of mean CRP levels by 20% or more was found in patients afteran MI. CRP levels are higher in survivors of MI with or without ademonstrable coronary lesion and increase further if other sites, suchas peripheral vasculature, also are involved. Hence, CRP levels mayserve to represent the inflammatory burden.

In the Monitoring Trends and Determinants in Cardiovascular Diseasetrial (Koenig W, Sund M, Fröhlich M, et al. C-reactive protein, asensitive marker of inflammation, predicts future risk of coronary heartdisease in initially healthy middle-aged men: results from the MONICA(Monitoring Trends and Determinants in Cardiovascular Disease) AugsburgCohort Study, 1984 to 1992. Circulation 1999; 99(2):237-42), a long-termprospective study of cardiovascular risk, patients with the highest CRPlevels had 2.6 times the risk of MI. In another study (Haverkate F,Thompson S G, Duckert F. Haemostasis factors in angina pectoris;relation to gender, age and acute-phase reaction. Results of the ECATAngina Pectoris Study Group. Thromb Haemost 1995; 73(4):561-7),postinfarction angina occurred in only 14% of patients with a normal CRPlevel. By comparison, 64% of patients admitted with high CRP levels hadevidence of postinfarction angina; nearly 42% requiredrevascularization, and 21% had recurrent MI.

In patients with MI, increased CRP concentration is associated with thepresence of complex angiographic lesions and the need forrevascularization (Moukarbel G V, Arnaout M S, Alam S E. C-reactiveprotein is a marker for a complex culprit lesion anatomy in unstableangina. Clin Cardiol 2001; 24(7):506-10). Elevated CRP levels also mayrepresent a biomarker for patients who are most susceptible toreocclusion. In patients with stable CAD who underwent stentimplantation following angioplasty, CRP levels increased over 96 hoursin those with restenosis; in patients without restenosis, CRP levelspeaked at 48 hours and then declined.

The normal serum concentration of CRP ranges from 3 mg/dL (90thpercentile of the general US population) to more than 200 mg/dL.Generally, the American Heart Association has suggested that if hs-CRPlevel is lower than 1.0 mg/L, a person has a low risk of developingcardiovascular disease; if hs-CRP is between 1.0 and 3.0 mg/L, a personhas an average risk, and if hs-CRP is higher than 3.0 mg/L, a person isat high risk for cardiovascular disease.

Because these ranges are not sensitive for the values required todetermine cardiovascular risk in otherwise healthy persons,investigators have developed new, modified techniques to measurehigh-sensitivity CRP. The high-sensitivity CRP assay has been shown todetect concentrations below 0.2 mg/mL and uses labeled monoclonal orpolyclonal anti-CRP antibodies in an enzyme-linked immunosorbent assay(ELISA) or an immunofluorescent assay (see, Rifai N, Ridker P M.High-sensitivity C-reactive protein: a novel and promising marker ofcoronary heart disease. Clin Chem 2001; 47(3):403-11).

Many conditions, activities, and medications affect levels of C-reactiveprotein. Increased levels are seen following allografts and graftocclusion, in connective tissue diseases (e.g., lupus erythematosus,Wegener's granulomatosis), arthritis, coronary artery disease, obesity,sepsis, in smokers, etc. Decreased levels of CRP are seen in response toinhibitory cytokines, exercise, and therapeutic doses of aspirin or3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (“statins”).Accordingly, the compositions of the present invention, whenadministered to a subject having inflammation or the related/resultantdisorders listed, can reduce CRP levels thereby reducing theinflammation and the severity of the related/resultant disorders.Administration of these compositions along with aspirin, naproxen,nambutome, other NSAID's or with statins appears to have a synergisticor additive effect on controlling inflammation and disease, depending onthe particular combination.

Atherosclerosis

Atherosclerosis remains the major cause of death and prematuredisability in developed societies. Moreover, current predictionsestimate that by the year 2020 cardiovascular diseases, notablyatherosclerosis will become the leading global cause of total diseaseburden, defined as the years subtracted from healthy life by disabilityor premature death. Atherosclerosis is an inflammatory vascular diseasecharacterized by endothelial activation, cellular influx, and productionof mediators and cytokines. This process leads to the formation of foamymacrophages and atheromatous plaques and, finally, to atherothromboticdisease. Atherosclerosis is associated with high morbidity andmortality.

Atherosclerosis is a complex process that leads to heart attack, stroke,and limb loss by the plugging of the arteries with atheroscleroticplaque. This plaque is a form of oxidized fat. It is now generallyrecognized that atherosclerosis is a chronic inflammatory disease,characterized by overrecruitment of leukocytes (monocytes and T-cells)to the site of inflammation. Vascular injury in response tocardiovascular risk factors promotes endothelial dysfunction, resultingin enhanced adhesion molecule expression and secretion ofpro-inflammatory cytokines and chemokines. This, in turn, leads toadherence, migration and accumulation of leukocytes withinatherosclerotic lesions. The recent findings on inflammatory processesinvolved in atherosclerosis development provide important links betweenrisk factors and the mechanisms of atherogenesis. Thus, researchinterest has increasingly focused on inflammatory biomarkers as means ofpredicting the risk of future clinical events. Indeed, elevated plasmalevels of molecules such as soluble intercellular adhesion molecule-1,interleukin-6 or C-reactive protein (CRP) have been shown to representinflammatory markers of future cardiovascular risk. Among these, CRP hasemerged as the most powerful and accessible for clinical use (see,Steffens S., Inflammation and Atherosclerosis, Herz. 2004 December;29(8):741-748).

CRP is a member of the pentraxin protein family, which is so namedbecause these proteins possess five identical subunits. CRP, which iselaborated dramatically during acute inflammation, augments the immuneresponse to certain antigens, activates complement, and increases themonocytic production of tissue factors. CRP binds to phosphoryl cholineon bacterial surfaces, acting as an opsonin and playing a pivotal rolein host defense. Interestingly, CRP also appears to bind low-densitylipoprotein cholesterol (LDL-C) in vitro, which suggests a directinteraction with the atherogenic lipids.

Inflammation in the vessels leads to the release of reactive oxides andradicals from the immune cells. When radicals react with lipids, theconsequence is lipid peroxidation. While a number of factors influencethe development and severity of atherosclerosis, a major factor is theROS-mediated peroxidation of serum low-density lipoproteins (LDLs). Thedietary approach to the prevention of heart disease and stroke is basedpartially on adding dietary antioxidants to limit LDL oxidation, as wellas decreasing the intake of fat itself. These approaches already havemade significant inroads into the mortality from heart disease, but thecompositions of the present invention may offer a safe pharmacologicalprevention in the future that is not as dependent upon willpower as arediet and exercise. The maintenance/increase of antioxidant potential byadministering the compositions of the present invention to a subject,therefore, can prevent or treat cardiovascular disease, e.g.,atherosclerosis.

Hypertension

The link between the elevation of the arterial blood pressure and theproduction of vascular lesions remains an area of inquiry. Recentevidence in the spontaneously hypertensive rat and the salt dependentDahl hypertensive strains indicate that there is an excessive productionof superoxide anion in microvascular endothelium and in circulatingleukocytes (Swei et al., Mechanisms of Oxygen Free Radical Formation inExperimental Forms of Hypertension, On-line Proceedings of the 5thInternet World Congress on Biomedical Sciences '98 at McMasterUniversity, Canada, Presentation # SAswei0837, 1998). These studiesexamining the role of the endothelial xanthine oxidase as a source forthe oxidative stress in the SHR and Dahl forms of hypertension and theirnormotensive controls indicated that both models of hypertension exhibitsignificantly elevated levels of XD and XO and enhanced oxidative stressin both the arterial and venular segments of the microcirculationcompared with their respective normotensive controls. The elevation ofthe oxidative stress and blood pressures could be reduced significantlyby blockade of XD and XO. Similarly, the maintenance/increase ofantioxidant potential by administering the compositions of the presentinvention to a subject, therefore, can prevent or treat cardiovasculardisease, e.g., hypertension.

Membrane abnormalities in essential hypertensives (EH) are known. Therespiratory burst enzyme, NADPH oxidase is located in the cell membraneof the neutrophil (PMNLs) and its activity is important in generation ofoxygen derived free radical (ROS). As noted above, ROS have beenimplicated in vascular changes in variety of conditions. Sagar andcoworkers studied the status of ROS and antioxidants in EH (Sagar etal., Mol Cell Biochem. 1992 April; 111(1-2):103-8).

Sagar and coworkers studied ten, age and sex-matched, healthy controls(GpI) and 26 untreated EH (Gp IIA mild-8, Gp IIB Moderate-8, Gp IICSevere-10). After clinical examination and basic laboratory evaluationof subjects, neutrophils isolated from their blood were studied.Chemiluminescence (CL) emitted by PMNLs after stimulation was measured(counts/min) in a luminometer and was taken as measure of OFR productionand thereby of NADPH oxidase activity. The levels of antioxidants,superoxide dismutase (SOD) and reduced glutathione (GSH), were alsoestimated. Chemiluminescence was increased significantly (p less than0.01) in Gp IIC (243.04+/−24.9×10(3) counts per minute) as compared toGp IIA (2.80+/−1.87), Gp IIB (34.54+/−30.24) and Gp I (0.52+/−0.15) andSOD was reduced significantly (p less than 0.05) in all EH (Gp IIA3.9+/−0.3 units per mg protein, Gp IEB 3.5+/−0.3 and Gp IIC 3.12+/−0.3)as compared to controls (4.1+/−0.2). Similarly GSH was reduced (p lessthan 0.05) in EH (Gp IIA 11.2+/−1.7 mg per gm protein, Gp IIB 8.5+/−1.1and Gp IIC 6.6+/−0.3) as compared to Gp I (13.5+/−2.5).

In addition, there is significant evidence that indicates blood vesselsthicken as a result of oxidative stress. Calcium antagonists, especiallythe highly lipophilic amlodipine, lacidipine and nisoldipine, are shownto possess antioxidant properties. These drugs reduce the oxidation ofLDL and its influx into the arterial wall, and reduce atheroscleroticlesions in animals. Platelet production of malondialdehyde, a marker ofoxygen free radical formation, is suppressed by amlodipine, lacidipineor nifedipine in hypertensive patients. In the Regression GrowthEvaluation Statin Study (REGRESS), co-administration of calciumantagonist, amlodipine or nifedipine with pravasatin caused asignificant reduction in the appearance of new angiographic lesions. Inthe Verapamil in Hypertension and Atherosclerosis Study (VHAS),verapamil was more effective than chlorthalidone in promoting regressionof thicker carotid lesions in parallel with a reduction in the incidenceof cardiovascular events. In the Prospective Randomized Evaluation ofthe Vascular Effects of Norvasc Trial (PREVENT), amlodipine slowed theprogression of early coronary atherosclerosis in patients with coronaryartery disease. In a subprotocol of the Intervention as a Goal in theHypertension Treatment (INSIGHT) study, nifedipine GITS significantlydecreased intima-media thickness as compared to co-amilozide(hydrochlorothiazide+amiloride). Preliminary results of the EuropeanLacidipine Study on Atherosclerosis (ELSA) show that lacidipine reducedthe intima-media thickness progression rate as compared to atenolol.Thus, selective calcium antagonists treat hypertension and are potentialantiatherosclerotic agents. (see, Hernandez, R. H., Calcium antagonistsand atherosclerosis protection in hypertension, Am J Ther. 2003November-December; 10(6):409-14). Likewise, the maintenance/increase ofantioxidant potential by administering the compositions of the presentinvention to a subject, alone or in conjunction with calcium channelantagonists therefore, can prevent or treat cardiovascular disease,e.g., atherosclerosis, arterial lesions, intima-media thickening, andhypertension. This is illustrated further in the examples below.

Arthritis

Rheumatoid arthritis (RA) is a chronic multisystem disease of unknownetiology. Although there are a variety of systemic manifestations, thecharacteristic feature of RA is persistent inflammatory synovitis,usually involving peripheral joints in a symmetric distribution. Thepotential of the synovial inflammation to cause cartilage destructionand bone erosions and subsequent changes in joint integrity is thehallmark of the disease.

Direct correlation between the increases in TNF-alpha and MMP-1production and collagen degradation seen in the RA patient suggests thatcollagenase cleavage of cartilage collagen is related to the activitiesof TNF-alpha and MMP-1. The reduction in cartilage type II collagensynthesis in early RA may contribute to the developing pathology, sincea lack of synthesis of this molecule would inhibit maintenance ofcartilage matrix. (see, Fraser A., Turnover of type II collagen andaggrecan in cartilage matrix at the onset of inflammatory arthritis inhumans: relationship to mediators of systemic and local inflammation.Arthritis Rheum. 2003 November; 48(11):3085-95).

Deterioration of the joint is less likely to occur when the patient CRPlevels are consistently controlled (see, Dawes, P. T., Rheumatoidarthritis: treatment which controls the C-reactive protein anderythrocyte sedimentation rate reduces radiological progression. TheBritish Journal of Rheumatology, Vol 25, 44-49). CRP levels may also behelpful in following response to therapy in rheumatic disorders, and mayhelp to differentiate rheumatoid arthritis (high levels of C-reactiveprotein) from uncomplicated lupus (low levels of C-reactive protein).When used to evaluate patients with arthritis, serum is the preferredspecimen; there is no reason to examine synovial fluid for C-reactiveprotein. CRP levels, insulin sensitivity, HDL cholesterol, triglyceridesand hypertension are inter-related in RA patients, who typicallyexperience a markedly increased frequency of cardiovascular disease(see, Dessein P H, Cardiovascular risk in rheumatoid arthritis versusosteoarthritis: acute phase response related decreased insulinsensitivity and high-density lipoprotein cholesterol as well asclustering of metabolic syndrome features in rheumatoid arthritis.Arthritis Res. 2002; 4(5):R5).

Generally, the first line of medical management of RA is the use ofnonsteroidal anti-inflammatory drugs (NSAIDs) and simple analgesics tocontrol the symptoms and signs of the local inflammatory process. Theseagents are rapidly effective at mitigating signs and symptoms, but theyappear to exert minimal effect on the progression of the disease. NSAIDsblock the activity of the Cox enzymes and therefore the production ofprostaglandins, prostacyclin, and thromboxanes. As a result, they haveanalgesic, anti-inflammatory, and antipyretic properties. In addition,the agents may exert other anti-inflammatory effects. Since these agentsare all associated with a wide spectrum of undesirable and even toxicside-effects, the natural dietary supplement compositions of the presentinvention provide a non-toxic alternative to NSAIDs.

Although osteoarthritis (OA) is thought to derive from defectivechondrocyte metabolism and thus inherently lack the large scale systemicresponse of rheumatoid arthritis, there is increasing interest in theacute phase proteins in OA (see, Sowers M., C-reactive protein as abiomarker of emergent osteoarthritis. Osteoarthritis Cartilage. 2002August; 10(8):595-601). Severity of pain, is associated with hsCRPlevels in patients with advanced OA (see, Sturmer T., Severity andextent of osteoarthritis and low grade systemic inflammation as assessedby high sensitivity C reactive protein, Ann Rheum Dis. 2004 February;63(2):200-5).

Accordingly, the maintenance/increase of antioxidant potential in amammal provides a method of ameliorating or decreasing tissuedegradation and inflammation seen in arthritis (RA and OA). Byadministering the compositions of the present invention to a subject asdescribed above, the compositions can therefore, prevent or treat OA andRA. The compositions may be given with other pharmaceutical agents,e.g., Relafen and other NSAID's or glucocorticoids (cortisone,dexamethasone, etc.) to achieve a greater anti-inflammatory effect.

Cancer and Other Malignancies

Cancer and other malignancies all entail unconstrained cell growth andproliferation based upon changes in the cell's genetic information. Inmost cases, for example, one or more genes that normally constrain cellgrowth and replication is/are mutated, or otherwise inactivated. Thesegenetic deficiencies correspond directly with deletions and sequencechanges in the genetic code, resident in the cell's DNA. A frequentlyseen final common cause of such DNA damage is free radical injury. Ofthe myriad injuries sustained by our DNA on a daily basis, most arerepaired by normal DNA repair mechanisms within the cell, while someresult in cell death. Since such injuries are sporadic and distributedsomewhat randomly across the genome, most lethal DNA injuries areclinically inconsequential, resulting in the loss of a few cells amongmillions. However, when a single cell sustains an injury that impairsgrowth regulation, it can proliferate disproportionately and growrapidly to dominate the cell population by positive natural selection.The result is a tumor, frequently a malignant one, where the constraintof growth and proliferation is particularly deficient. Therefore, freeradical injury to the genetic material is a major final common pathwayfor carcinogenesis.

An approach to creating anti-tumor therapeutics that appears to haveearly success is aimed at preventing tumor induced angiogenesis, therebyreducing blood supply to the tumor to prevent growth and to kill theproliferating cells by starving them of nutrients and oxygen. While mostcandidate therapeutics are direct inhibitors of angiogenesis, othertreatments are designed to prevent initiation of the angiogenesisresponse. Tumor formation is associated with localized inflammation, andincreases of c-reactive proteins (CRP), which are well known prognosticindicators of patient survival (see, McKeown, D J., The relationshipbetween circulating concentrations of C-reactive protein, inflammatorycytokines and cytokine receptors in patients with non-small-cell lungcancer. Br J Cancer. 2004 Dec. 13; 91(12):1993-5). Elevated CRP levelsare seen in many types of cancer, see for example, McArdle P A Therelationship between interleukin-6 and C-reactive protein in patientswith benign and malignant prostate disease, Br J Cancer. 2004 Nov. 15;91(10):1755-7; Saddler, D., C-reactive protein elevation and the risk ofcolorectal cancer, Gastroenterol Nurs. 2004 September-October;27(5):246-7; and Alexandrakis, MG The relation between bone marrowangiogenesis and the proliferation index Ki-67 in multiple myeloma, JClin Pathol. 2004 August; 57(8):856-60.

CRP can significantly influence gene expression in the vascularendothelium. CRP is upregulated by IL-6, and increases expression ofIL-8, ZF9, Activin A, MCP-1, EXT1, Cited2, PAI-1, Fibronectin-1, Gravin,Connexin-43, and SORL-1, and decreases expression of MAT2A, WRB, RCN1,TEB4, DNCL1 and Annexin A1 (see, Wang, Q., Effect of C-Reactive Proteinon Gene Expression in Vascular Endothelial Cells, Am J Physiol HeartCirc Physiol. 2004 Dec. 9). Thus, CRP-responsive genes may have a broadfunctional role in cell growth and differentiation, vascular remodelingand solid tumor development. Inhibiting CRP would provide an additionalapproach to current cancer therapies and may provide a prophylacticanticancer effect by inhibiting inflammation and angiogenesis.

By administering the compositions of the present invention to a subjectas described above, the compositions can therefore, prevent or treatcancers that respond to antioxidant treatments. Such cancers include forexample but not limited to, breast cancer (see, Kline K, Vitamin E andbreast cancer, J Nutr. 2004 December; 134(12 Suppl):3458S-3462S); lungcancer (see, Wright M E, Development of a comprehensive dietaryantioxidant index and application to lung cancer risk in a cohort ofmale smokers. Am J Epidemiol. 2004 Jul. 1; 160(1):68-76); ovarian cancer(see, Anderson K, Differential response of human ovarian cancer cells toinduction of apoptosis by vitamin E Succinate and vitamin E analogue,alpha-TEA. Cancer Res. 2004 Jun. 15; 64(12):4263-9); and colon cancer(see, Al-Shaer M H., C-reactive protein and risk of colon cancer. JAMA.2004 Jun. 16; 291(23):2819). In fact, colorectal carcinogenesis isassociated with serious oxidative stress gradual advancement ofoxidative-antioxidative disorders is followed by progression ofcolorectal cancer (see, Skrzydlewska E, Lipid peroxidation andantioxidant status in colorectal cancer. World J Gastroenterol. 2005Jan. 21; 11(3):403-6).

The compositions may be given with other pharmaceutical agents, e.g.,glucocorticoids, camptothecins, mustard agents, and other chemotherapydrugs, to achieve a greater anti-tumor response and to controlinflammation.

Radiation Injury

Radiation injury represents an important cause of ROS-mediated disease.With respect to commonly encountered levels of radiation, depending uponthe situation, about two-thirds of the sustained injury is mediated notby the radiation itself, but by the ROS generated secondarily. Thisapplies not only to the acutely toxic forms of radiation injury, but thelong-term, mutagenic (and hence carcinogenic) effects as well.

An important clinical application of this principle is encounteredregularly in the treatment of cancer by radiation therapy. Large tumorsoften outgrow their blood supplies and tumor cells die within thecenter, despite being well oxygenated at the periphery. Between thesetwo regions is an area of tumor that is poorly oxygenated, yet remainsviable. Radiation therapy of such tumors is particularly effective atthe periphery, where an abundant concentration of oxygen is available toform tumorcidal ROS. The poorly oxygenated center is injured to asignificantly smaller degree. While the dead cells in the center don'tsurvive anyway, the poorly oxygenated, yet viable, cells between thesetwo areas can survive a safe dose of radiation therapy, and thereby seeda later local recurrence of the tumor. This is a major reason why manylarge tumors are treated by a combination of radiation therapy (to killthe tumor at its advancing edges) and surgical removal of the bulk ofthe tumor, including these particularly dangerous remaining cells.

ROS can be generated within the cell not only by external sources ofradiation, but also within the body as a by-product of normal metabolicprocesses. An important source of endogenous free radicals is themetabolism of some drugs, pollutants, and other chemicals and toxins,collectively termed xenobiotics. While some of these are directly toxic,many others generate massive free radical fluxes via the very metabolicprocesses that the body uses to detoxify them. One example is themetabolism of the herbicide paraquat. At one time, drug enforcementauthorities used this herbicide to kill marijuana plants. Growersrealized they could harvest the sprayed crop before it wilted, and stillsell the paraquat-laced product. Many who smoked this productsubsequently died of a fulminate lung injury. Fortunately, this approachhas been abandoned as a particularly inhumane way to solve the drugproblem.

While the paraquat story is a particularly striking example of ametabolic mechanism of free radical toxicity, many commonly encounteredxenobiotics, including cigarette smoke, air pollutants, and even alcoholare toxic, and often carcinogenic to a large degree by virtue of thefree radicals generated by their catabolism within our bodies. Moreover,there is accumulating evidence that a diet rich in fruits andvegetables, which are high in natural antioxidants, and low in saturatedfat (a particularly vulnerable target for damage by ROS), reduces therisk of atherosclerosis and cancer. The maintenance/increase ofantioxidant potential by administering the compositions of the presentinvention, therefore, can prevent or treat atherosclerosis and cancer.

The maintenance/increase of antioxidant potential by administering thecompositions of the present invention to a subject, therefore, canprevent or treat radiation-mediated injury.

Neurological and Neurodegenerative Diseases

Neurological and neurodegenerative diseases affect millions ofAmericans. These include depression, obsessive-compulsive disorder,Alzheimer's, allergies, anorexia, schizophrenia, as well as otherneurological conditions resulting from improper modulation ofneurotransmitter levels or improper modulation of immune systemfunctions, as well as behavioral disorders such as ADD (AttentionDeficit Disorder) and ADHD (Attention Deficit Hyperactivity Disorder).Oxidative stress links diverse neuropathological conditions that includestroke, Parkinson's Disease, and Alzheimer's Disease and has beenmodelled in vitro with various paradigms that lead to neuronal celldeath following the increased accumulation of reactive oxygen species.For example, immortalized neurons and immature primary cortical neuronsundergo cell death in response to depletion of the anti-oxidantglutathione, which can be elicited by administration of glutamate athigh concentrations.

A number of these diseases appear to have ROS toxicity as a centralcomponent of their underlying mechanism of nerve cell destruction,including, but not limited to, amyotrophic lateral sclerosis (ALS, orLou Gehrig's disease), Parkinson's disease, and Alzheimer's disease. Forexample, Alzheimer's disease is a neurodegenerative disorder associatedwith aging and cognitive decline. Amyloid beta peptide (1-42) is aprimary constituent of senile plaques—a hallmark of Alzheimer'sdisease—and has been implicated in the pathogenesis of the disease.Studies have shown that methionine residue 35 of beta(1-42) may play acritical role in Abeta(1-42)-mediated oxidative stress and neurotoxicity(see, Boyd-Kimball D, Rodent Abeta(1-42) exhibits oxidative stressproperties similar to those of human Abeta(1-42): Implications forproposed mechanisms of toxicity. J Alzheimer's Dis. 2004 October;6(5):515-25).

Additionally, oxidative stress is associated with the selective loss ofdopaminergic neurons of the substantia nigra in Parkinson's disease. Therole of alpha synuclein as a potential target of intracellular oxidantshas been demonstrated by identification of posttranslationalmodifications of synuclein within intracellular aggregates thataccumulate in PD brains, as well as the ability of a number of oxidativeinsults to induce synuclein oligomerization (see, Cole NB.,Metal-catalyzed oxidation of alpha synuclein: helping to define therelationship between oligomers, protofilaments and filaments. J BiolChem. 2004 Dec. 21).

Accordingly, the maintenance/increase of antioxidant potential byadministering the compositions of the present invention to a subject,therefore, can prevent or treat neurological and neurodegenerativediseases that have inflammation and oxidative stress as causative orcomplicating factors.

Ischemia/Reperfusion Injury

When an organ is deprived of its blood supply (ischemia) it is injured,not just by the temporary loss of oxygen, but also by the ROS that aregenerated by reaction with the oxygen that is reintroduced atreperfusion, when the blood supply is restored. In some clinicalsituations, this injury can prevented by giving antioxidants, sometimeseven after the period of ischemia, but just prior to reperfusion. Forexample, the preservation of kidneys, livers, and other organs insolutions that contain antioxidants, as well as other agents, is nowroutine prior to their transplantation. Another example is the use ofdrugs that block the function of free radical generating enzymes priorto stopping the heart for cardiac surgery. These drugs help preventreperfusion injury when the heart is restarted and flow is restored.This reperfusion injury mechanism also has been found to play animportant role in patients suffering from multiple organ failure aftertrauma, massive surgery, or shock. Multiple organ failure is now theleading cause of death in intensive care units, and extensive effortsare under way to understand better how ROS contribute to this syndrome.

Ischemia, which is low tissue oxygen saturation of a given tissue, canoccur in any organ system. All organs require a blood supply in order toremain viable. The intact organ whose arterial supply is compromised(either by partial or total occlusion) is rendered ischemic (e.g.,coronary artery occlusion, organs awaiting transplantation, cerebralvascular accident, compartment syndrome, etc.). There are reversible andirreversible histological, physiological and biochemical changes whichoccur as a result of ischemic injury to tissue. End stage ischemia isuniversal and demonstrates necrosis. Demopoulos et al. (Fed. Proc.32:1859-1861, 1973b) theorized that the necrosis observed in ischemictissue was due to oxidants generated by the uncoupling of the oxidativephosphorylation chain in mitochondria. Zweir, et al., provided directevidence that free radical production resulted from ischemia usingelectron spin resonance spectroscopy (Proc. Natl. Acad. Sci. USA, vol.84, pp: 1404-1407). In reperfusion studies Zweir showed the alterationof one of the free radicals with the use of superoxide dismutase (whicheliminated superoxide). In ischemic cardiac myocyte a depletion of ATPinduces the release of arachidonic acid and palmitic acid. Vitamin E(Massey, K. D. and Burton, K. P.: Am. J. Physiol. 256 (Heart Circ.Physiol. 25): H1192-H1199, 1989), vitamin E acetate and seleniumselenite have been used to protect tissue against free radicals, whichhave occurred in ischemia. It is postulated that enhancement of tissueantioxidants would eliminate the superoxide free radical, as well asother oxidants that are not double produced as a result of ischemia andprostaglandin metabolite production. The maintenance/increase ofantioxidant potential by administering the compositions of the presentinvention to a subject, therefore, can prevent or treatischemia/reperfusion injury, e.g., brain ischemia and heart ischemia(i.e., myocardial infarction).

Aging

Aging is a remarkably complex process that has managed to remainrelatively opaque to scientific understanding. There is now evidencethat aging is a series of processes, i.e., a series of controlledmechanisms, and not just the passive accumulation of wear and tear overthe years. If aging is a series of processes, some of these processesare potentially controllable, or at least modifiable. One of the mostimportant of these processes is comprised of an accumulation of themolecular injuries that are mediated by free radicals and other ROS.Recent studies indicate that the therapeutic manipulation of ROSmetabolism can actually extend the total life span of mice to asignificant degree. The maintenance/increase of antioxidant potential byadministering the compositions of the present invention to a subjectcan, therefore, prevent or treat age-mediate injury.

Burns/Wound Healing

Burn wounds to skin and other organs can occur by ultraviolet radiation(UV), chemical agents, conductive or convective heat, electrocution,etc. Burns can occur in lung parenchyma by the inhalation of smoke orcaustic gases (see section on tissue injury). Burn wounds to the skinare graded as first, second, and third degree burns (the most severe).It is postulated that any burn wound produces tissue damage, largely bythe production of oxidants (Till, G. O.: Am J. Pathol. July; 1325(1):195-202, 1989). Liposomes (artificial membranes) when exposed to UVundergo peroxidation (Bose, B: Biotechnol Appl. Biochem, Oct., 12 (5):557-61, 1990). It has been postulated that similar peroxidation occursin skin when it is exposed to UV radiation (Somer, E.:Shape Magazine, p33-35, November 1992; Hamanka, H.: J. Dermatol, Oct. 17(10):595-8,1990). Exposure of skin to UV varies in intensity and length ofexposure. Daily exposure to UV (e.g., sunlight) has been postulated toresult in skin wrinkling. Depending on the intensity and/or length ofskin exposure to UV light, first, second or third degree burns canresult.

Hairless mice exposed to a single exposure of UV resulted in a broadrange decrease of antioxidants: glutathione, beta-carotene,alpha-tocopherol. The enzyme activity of catalase and glutathionereductase was also decrease (Fuchs, J.: J. Invest Dermatol, Dec., 93(6):769-73, 1989). These decreases in the concentration of antioxidants andenzyme activity in skin due to UV exposure supports the concept of theoccurrence of free radicals in skin. It is postulated that lipidperoxidation could be inhibited by an enhancement of antioxidants inskin. Lipid peroxidation in liposomes exposed to UV can be inhibited byplacement of beta-carotene or alpha-tocopherol in liposomes (Pelle, E.:Arch. Biochem. Biophys. Dec. 283 (2): 234-40, 1990).

Excessive free radical production has been cited as a factor in delayedwound healing (Yukie, N.: Dermatolgica, 179 (suppl 1): 101-106, 1989).The maintenance/increase of antioxidant potential by administering thecompositions of the present invention to a subject of antioxidantswould, therefore, ameliorate the effects of pathologic oxidants andprostaglandin production as well as promote wound healing in variousskin injuries.

Tissue Injury and Degeneration

Tissue injury occurs as a result of an inflammatory focus occurring inthe area of a cell or an organ. For example plasma oxidative stressoccurs in patients with ulcerative colitis, and omega-3 fatty acids areunder study as free radical scavengers for protecting the patientsagainst the overall effect of oxidative stress (see, Barbosa D S,Decreased oxidative stress in patients with ulcerative colitissupplemented with fish oil omega-3 fatty acids., Nutrition. 2003October; 19(10):837-42). C-reactive protein levels corresponded closelywith clinical and pathological indices of relapse, remission andresponse to therapy in patients with Crohn's disease. Assay of serumC-reactive protein provides an objective criterion of inflammatoryactivity, which may be useful in the assessment, management and study ofCrohn's disease (see, Fagan, E. A., Serum levels of C-reactive proteinin Crohn's disease and ulcerative colitis. Eur J Clin Invest. 1982August; 12(4):351-9). Accordingly, CRP levels may also be helpful infollowing response to therapy for tissue injuries, and may help todifferentiate in Crohn's disease (high C-reactive protein) fromulcerative colitis (low C-reactive protein).

Inflammation can occur due to a local inducement (e.g. hepatitis) or dueto an injury occurring to one organ in a remote location and anotherdiscontiguous organ, which also sustains an injury (e.g., severe burnsoccurring to skin (the first organ) with subsequent injury to the lungs(the second organ)). In either case, local or remote tissue injury isbelieved to be mediated by activated leukocytes, which release oxidants.Oxidants released from leukocytes react with cellular (organ) membranes(Fantone, J. C. and Ward, P. A.: Am. J. of Path., vol. 107(3), P.397-418, 1982). Repeated cellular membrane exposure to oxidantsdecreases antioxidant levels, which increases their susceptibility todamage. Increasing the levels of antioxidants in the extracellularand/or intracellular, and/or the lipid-aqueous interface is postulatedto thwart oxidant damage to vital cellular structures. Themaintenance/increase of antioxidant potential by administering thecompositions of the invention to a subject can, therefore, prevent ortreat ROS-mediated tissue injury or other types of inflammatory tissuedegeneration.

Sepsis

Sepsis is characterized as a systemic infection by a microorganism.Frequently it is fatal and if not fatal increases the morbidity of thepatient. In sepsis, red blood cells become sticky and deformed (Baker,C. H., et al.: Circ. Shock 20:127-139, 1986; Powell, J., et al.:Critical Care Med., vol 19 (5), 1991), which can lead to occlusion ofthe microvasculature. Cardiac output is increased, but in the kidney,liver, and musculature blood flow is decreased (Hurd, T. C., et al.:Archives of Surg., vol. 123, 1988). Evidence of free radical damage hasbeen demonstrated in in vitro and in vivo studies involving shockinduced by endotoxins (McKechnie, K., et al.: Circ. Shock 19: 429-439,1986). Findings include increased vascular permeability, damagedmitochondria, disruption of calcium transport by the sarcoplasmicreticulum, and the activation of the complement system (particularlyC5a). In septic infections, serum levels of acute phase proteins,particularly CRP, are elevated, which increases activation of thecomplement response as well as other cell mediated responses.

The maintenance/increase of antioxidant potential by administration ofthe compositions of the present invention to a subject can, therefore,prevent or treat sepsis-mediated inflammatory cellular damage.

GSH Deficiency

Artificial depletion of glutathione interferes with normal T cellfunction, particularly within the first 30-60 minutes of activation(Fischman, C. M., et. al: The Journal of Immunology, vol. 127(6), p2257-2262, 1981; Hamilos, D. L. and Wedner, H. H.: Journal ofImmunology, vol. 135 (4), 1985). Glutathione deficient T cells showed adecrease in thymidine incorporation and blast transformation. Thegreater the depletion of glutathione the longer it took cells to recoverto normal levels. If cellular GSH depletion was severe enough the cellsnever recovered to normal GSH levels. Increased glutamate levels, whichare found in AIDS patients (Eck, H.-P. and Droge, W.: Bio. Chem.Hoppe-Seyler, vol. 370, pp 109-113), appear to inhibit the transport ofcystine into macrophages. Under normal circumstances cysteine is reducedto cysteine by the macrophages. Cysteine is exported into themicroenvironment for the use of T cells for the ultimate conversion tointracellular glutathione. T cells cannot utilize cystine. In AIDSpatients glutathione is depleted (Eck, H.-P, et al.: Biol. Chem.Hoppe-Seyler, vol. 370, pp 101-108), which is postulated to adverselyeffect T cell function. This scenario is believed to be similar to theexperimental studies, which demonstrated abnormal T cell function as aresult of artificial GSH depletion. The maintenance/increase ofantioxidant potential by administering the compositions of the presentinvention to a subject can, therefore, prevent or treat GSH deficiency.

AIDS

There is considerable evidence which indicates that HIV infection andsubsequently ARC/AIDS is by in large a free radically mediated disease.This analysis can be made indirectly as judged by the antioxidant levelsin humans and their consequences on the immune system. One of thoseantioxidants, glutathione (GSH), is decreased as a result of HIVinfecting the host. The GSH levels continue to decrease as the diseaseprogresses through ARC and finally to AIDS. Micromolar changes in GSHlevels have an untoward effect on the function of T lymphocytes (whichcan be viewed as the pivotal leader of the immune system). GSH shows amultiplicity of uses in the immune system. Thiol concentrations (e.g.GSH) regulate the replication of HIV genomic expression (Kalebic, T., etal.: Proc. Natl. Acad. Sci., USA; 88: 986-90, 1991; Roeder, M., et al.,:Porc. Natl. Acad. Sci., USA, vol. 87, p 4884-4888). Increasing theconcentrations of thiols (GSH, NAC, GSE (glutathione ester)) in culturemedium of U1 cell line (promonocytes) results in suppression of viralassembly, HIV reverse transcriptase production and viral replication.The maintenance of antioxidant potential by administering thecompositions of the invention to a subject can, therefore, prevent ortreat HIV-mediated injury.

Immunomodulation

There are numerous activators of leukocytes (e.g., exposure toingestible particles, certain soluble factors such as complement,lectins, phorbol esters, etc.). A consequence of leukocyte activation isthe release of the MPO system (H₂O₂+halide+myleoperoxidase) and otheroxidants. The more potent the stimulus of activation of leukocytes is,the greater the release of oxidants and the greater the suppression of Tlymphocytic function. When activated leukocytes were combined with Tlymphocytes and catalase, there was no suppression of lymphocyticfunction; monocytes (which contain enzymatic antioxidants: glutathioneperoxidase, catalase, myeloperoxidase) were used in lieu of catalase,again there was no suppression (Lipsky, P. E.: J. Clin, Invest. 73:53,1984). Antibody production by B lymphocytes showed a similarsusceptibility to free radical damage as did lymphocytes (El-Hag, A., etal.: J. of Immunol., vol. 136 (9), 1986). A following is a rank orderfor various lymphocytic functions to free radical attack: immunoglobulinsecreting cells were the most sensitive (particularly to the MPOsystem); Natural Killer cell activity, DNA synthetic responses to PHAand Con A were intermediate; and the DNA response to PWM was the leastsusceptible. Monocytes/macrophages have approximately 15-20 times highercatalase content in comparison to lymphocytes (Meerhof, L. J. and Roos,D.: J. Reticulendothel. Soc. 28: 419) and would therefore be much lesssusceptible to oxidative damage. Lymphocytes exposed to a free radicalgenerating system demonstrate changes in membrane characteristics: 63%decrease in E rosette formation, 44% decrease in surface immunoglobulinsand 90% decrease in cap formation (Kraut, E. H. and Sagone, A. L.: J. ofLab. Clin. Med., November 1981, p 697-703). The maintenance/increase ofantioxidant potential by administering the compositions of the inventionto a subject can, therefore, prevent or treat immune damage by ROS.

Sickle Cell Anemia

Sickle cell anemia is a genetically determined disease. Analysis ofsickle cell patients RBC (HbS) demonstrates a number of peculiarities ofthe membrane: frozen spectrin shell of irreversibly sickled RBC, anabnormal orientation of the lipid bilayer phospholipids, deficientcalcium-ATPase, a propensity for HbS RBCs to adhere to vascularendothelium, and oxidized thiol groups on the HbS molecule. It is thecharacteristic of the tendency of adherence to the vascular endothelium,which is the likely primary pathogenesis of the disease, which isocclusion of the microvasculature. Consequently, ischemic injury occursto organs (see section on ischemia). Additional evidence of free radicaldamage to HbS is a deficiency of alpha-tocopherol, increased amounts ofmalondialdehyde, and abnormal group cross linking by malonadehyde.Superoxide anion can enter into erthrocytes via anion channels,resulting in the formation of methemoglobin and the ultimate lysis oferythrocytes (Weiss, S. J.: J. Biol. Chem. 225: 9912-9917, 1980). SickleRBCs spontaneously generate sixty percent greater quantities ofsuperoxide and approximately 75% more hydrogen peroxide when comparedwith controls (Hebbel, R. P., et al.: J. Clin. Invest., vol. 70, p.1253-1259, 1982). Superoxide dismutase is increased by about 50%,glutathione peroxidase and catalase were decreased by approximately 50%and 29% respectively. Glutathione and vitamin E levels weresignificantly reduced. It is postulated that by increasing both bonenarrow and serum antioxidant levels that free radicals produced bysickled RBCs would be markedly reduced. Accordingly,maintenance/increase of antioxidant potential by administering thecompositions of the present invention is useful to prevent or treatanemia-mediated injury.

Diabetes

Diabetes mellitus (DM) is a common disease affecting over 124 millionindividuals worldwide. DM is associated with high risk ofatherosclerosis and renal, neural, and ocular damage. Oxidative stressresults from a cell or tissue failing to detoxify the free radicals thatare produced during metabolic activity. Diabetes is characterized bychronic hyperglycemia that produces dysregulation of cellularmetabolism. Vincent and coworkers have suggested that diabetes overloadsglucose metabolic pathways, resulting in excess free radical productionand oxidative stress (Vincent et al., Endocr Rev. August; 25(4):612-28(2004). Vincent and coworkers have presented evidence to support theidea that both chronic and acute hyperglycemia cause oxidative stress inthe peripheral nervous system that can promote the development ofdiabetic neuropathy. Proteins that are damaged by oxidative stress havedecreased biological activity leading to loss of energy metabolism, cellsignaling, transport, and, ultimately, to cell death. Examination of thedata from animal and cell culture models of diabetes, as well asclinical trials of antioxidants, strongly implicateshyperglycemia-induced oxidative stress in diabetic neuropathy. Vincentet al., concluded that superior antioxidative therapies remainsessential for the prevention of neuropathy in diabetic patients (Vincentet al., Endocr Rev., August; 25(4):612-28 (2004). Themaintenance/increase of antioxidant potential by administering thecompositions of the invention to a subject can, therefore, prevent ortreat diabetes-related, ROS-mediated tissue damage.

Pasaoglu and coworkers, investigated lipid peroxidation, resistance ofplasma and red blood cells to oxidation, and antioxidant defense systemin erythrocytes and sera in patients with type 2 diabetes mellitus(Pasaoglu et al., Tohoku J Exp Med., July; 203(3):211-8 (2004). Onegroup included newly diagnosed 20 patients and the other included 20patients treated with oral antidiabetic agents (OAD). Twenty healthysubjects served as controls. Serum and red blood cell malondialdehyde(MDA), glutathione (GSH), resistance to oxidation, and plasma thiol(total —SH) levels were measured. In addition, glycated hemoglobin,serum fructosamine, uric acid, total protein, total cholesterol,triglyceride and glucose levels were determined. Although newlydiagnosed patients had higher serum and erythrocyte MDA levels thanthose of controls, the highest levels of MDA were determined in patientstreated with OAD. MDA levels after exposing to oxidation increased inOAD group more than in newly diagnosed patients. Total —SH anderythrocyte GSH levels of the both diabetic groups were lower thancontrols. These results showed that serum and erythrocyte lipidperoxidation was increased in diabetic patients. The sera of thepatients showed a decreased resistance against oxidation. Pasaoglu andcoworkers proposed that the effect of increased free radicals may beprevented by antioxidant systems in early stages of type 2 diabetes butin advanced stages this relationship is impaired owing to decreasedantioxidant activity. Decreased red blood cell GSH and serum total —SHlevels may be due to a compensation mechanism of the antioxidants. Themaintenance/increase of antioxidant potential by administering thecompositions of the invention to a subject can, therefore, prevent ortreat diabetes-related (e.g., diabetes type-2), ROS-mediated tissuedamage.

Administration of the antioxidant enzymes superoxide dismutase (SOD) andcatalase prevented destruction of islet allograffs in NOD mice (Nomikoset al., Immunol Cell Biol 67:85-87 (1989)). Furthermore, the antioxidantprobucol was shown to reduce the diabetes incidence and to delaydiabetes onset in the BB rats (Drash A L et al., Am J Cardiol, 62:27B-30B (1988)). Tabatabaie and coworkers have demonstrated that chronicadministration of the free radical scavenger phenyl-N-tert-butylnitrone(PBN) inhibits STZ-induced diabetes in mice (Tabatabaie et al., FEBSLett, 407:148-152 (1997)). The low level of antioxidant enzymes such asSOD, catalase, and glutathione peroxidase in the islets is anotherindication that β-cells are exceptionally vulnerable to oxidative damage(Lenzen et al., Free Radical Biol. Med., 20:463-466 (1996)).

ROS generation, evidenced by the formation of lipid peroxidationproducts, is believed to be the ultimate cause of cytokine-mediateddeath of β-cells in isolated islets (Rabinovitch et al., J. Clin.Endocrinol. Metab., 81:3197-3202 (1996)). Tabatabaie and coworkersrecently demonstrate the formation of free radicals in the pancreaticislets as a result of cytokine treatment using EPR spectroscopy(Tabatabaie et al., Diabetes, August (2003)). Based on their studies,Tabatabaie and coworkers concluded that free radicals have a role in thepathogenesis of type 1 diabetes through β-cell cytokine-mediated freeradical generation in the pancreatic Islets (Tabatabaie et al.,Diabetes, August (2003)). The maintenance/increase of antioxidantpotential by administering the compositions of the invention to asubject can, therefore, prevent or treat diabetes-related (e.g.,diabetes type-1), ROS-mediated tissue damage.

Pharmaceutical Compositions and Formulations

The compositions of the present invention can be used in beverages,tonics, infusions, or foodstuffs alone, or in combination with otherdietary supplements or therapeutics. The compositions of the inventioncan be used alone or further formulated with pharmaceutically acceptablecompounds, vehicles, excipients or adjuvants with a favorable deliveryprofile, i.e., suitable for delivery to a subject. Such compositionstypically comprise the compositions of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal compounds,isotonic and absorption delaying compounds, and the like, compatiblewith pharmaceutical administration. Suitable carriers are described inthe most recent edition of Remington's Pharmaceutical Sciences, astandard reference text in the field, which is incorporated herein byreference. Preferred examples of such carriers or diluents include, butare not limited to, water, saline, Ringer's solutions, dextrosesolution, and 5% human serum albumin. Liposomes and non-aqueous vehiclessuch as fixed oils may also be used. The use of such media and compoundsfor pharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or compound is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include oral, intravenous, intraperitoneal,subcutaneous, intramuscular, intraarticular, intraarterial,intracerebral, intracerebellar, intrabronchial, intrathecal, topical,and aerosol route. The pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide. Oral compositions generallyinclude an inert diluent or an edible carrier. They can be enclosed ingelatin capsules, caplets or compressed into tablets. For the purpose oforal therapeutic administration, the compositions of the invention canbe incorporated with one or more excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding compounds, and/oradjuvant materials can be included as part of the composition. Thetablets, pills, capsules, troches and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating compound such as alginicacid, Primogel, or corn starch; a lubricant such as magnesium stearateor Sterotes; a glidant such as colloidal silicon dioxide; a sweeteningcompound such as sucrose or saccharin; or a flavoring compound such aspeppermint, methyl salicylate, or orange flavoring. Excipients can alsoinclude, but are not limited to, e.g., calcium carbonate; croscarmellosesodium; dicalcium phosphate; magnesium stearate; microcrystallinecellulose; modified cellulose; silica; and stearic acid.

The compositions of the invention can also be prepared as pharmaceuticalcompositions in the form of suppositories (e.g., with conventionalsuppository bases such as cocoa butter and other glycerides) orretention enemas for rectal delivery.

In one embodiment, the compositions of the invention are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensionscan also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the composition of theinvention and the particular therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an active compoundfor the treatment of individuals. The pharmaceutical compositions can beincluded in a container, pack, or dispenser together with instructionsfor administration.

The present compositions may be provided as a single dose, however, itis preferred that the compounds be administered in multiple doses.Particularly preferred are intermittent doses, such as twice daily oronce daily doses. However beneficial effects are still seen with moresporadic intermittent doses, e.g., once every 36, 48, 60 or 72 hours, oronce per week. Duration of treatment will depend on the disorder beingtreated. For example, as discussed herein, an average 24% decrease ofserum CRP levels was observed in human subjects treated intermittentlyfor a 30 day period with the present compositions. Further decrease inserum CRP levels was observed to continue during the 90 day period ofthe study. Extrapolating this information to a suggested dosage regimen,one week may be sufficient to treat inflammation in post operativesurgical patients, whose serum CRP levels usually peak in three daysfollow surgery, and naturally decline shortly thereafter. Alternatively,chronic conditions such as arthritis or atherosclerosis may require longterm therapy over many years. A medical professional will be able todetermine dosage levels and intervals in view of the teachings providedherein.

The invention is further defined by reference to the following examples,which are not meant to limit the scope of the present invention. It willbe apparent to those skilled in the art that many modifications, both tothe materials and methods, may be practiced without departing from thepurpose and interest of the invention.

EXAMPLES Example 1 Herbal Compositions of the Invention AlterAntioxidant Enzymes and Decrease Lipid Peroxidation in Wild TypeC57BL/6J Mice I. General Purpose and Study Design

The purpose of this study was to observe and effects of an herbalcomposition of the present invention on normal 4 week old wild typeC57BL/6J mice. The exemplary herbal composition, Protandim I, is adietary supplement containing eight herbal extracts, including, B.monniera extract, Milk Thistle extract 70-80%, Ashwagandha powder,Turmeric extract 95% curcumin, Gotu kola powder, Aloe vera powder, Greentea (98% Polyphenols, 45% EGCG), and Ginko biloba leaf extract andspecifically detailed below in Table 3.

TABLE 3 Composition of Protandim I. Percent Weight Total ActiveIngredient Amount Active Ingredients Bacopa monniera extract, 45% 150 mg12.77 bacosides Milk Thistle extract, 70-80% 275 mg 23.4 silymarinAshwagandha powder 150 mg 12.77 Gotu kola power 150 mg 12.77 Turmericextract, 95% curcumin 75 mg 6.38 Green tea 98% polyphenols, 45% 75 mg6.38 EGCG Ginko biloba leaf extract 150 mg 12.77 Aloe vera powder 150 mg12.77 Total 1175 mg 100

The composition was mixed with the powdered diet, then pelleted. Micewere assigned to four groups as defined below in Table 4, receivingdosages ranging from 0 to 1, 3, or 10 times the anticipated humandosage. The estimated human dose for the present studies was 1175 mg/dayor 16 mg/kgbw/day. However, in other embodiments of the invention thedosage to a subject is the amount required to yield the desiredantioxidant effect.

TABLE 4 Mice received the indicated amounts of Protandim I DoseProtandim I No. of mice Diet μg/gbw/day mg/kg diet Group 1 7 Control 0 0Group 2 8 1X 16 100 Group 3 7 3X 48 300 Group 4 8 10X  160 1000 TOTAL 30

The amount added was based on the assumption that the average mouseconsumes 5 g/d. The amount of Protandim I composition received by eachmouse in Group 2 was equivalent in mg/gbw to the anticipated humandosage for a 70 kg person. Group 3 received a 3-fold higher dosage ofProtandim I and Group 4 received a 10-fold higher dosage of Protandim I.Mice were weighed at day 1 and day 23. After 23 days, the animals weresacrificed and tissues were harvested for analysis. The end pointsmeasured included activities of the major antioxidant enzymes SOD, CAT,and GPX in RBCs, liver, and brain. In addition, the extent of lipidperoxidation was assessed by measuring thiobarbituric acid reactivesubstances (TBARS) in plasma, liver, and brain.

Thiobarbituric acid reactive substances, or TBARS, were determined bythe method of Ohkawa et al. (Ohkawa et al., Anal. Biochem., 95: 351-358(1979). The reaction mixture (total volume of 1 ml) contained 50 μl of8.1% sodium dodecyl sulfate, 0.375 ml of 20% acetic acid and 0.375 ml0.8% thiobarbituric acid, and 200 μl of plasma or tissue homogenatesupernate. The mixture was heated in boiling water for 1 hr, cooled withtap water and extracted with n-butanol/pyridine (15:1 v/v) by vortexingfor 1-2 min. The mixture was then centrifuged at 500-1000 g for 10 minor until a good aqueous-organic phase separation occurred. The organicphase was removed and its absorbance at 532 nm was measured against areaction mixture blank. A standard curve was prepared with1,1,3,3-tetramethoxypropane, and TBARS are reported as molarequivalents.

Superoxide dismutase activity was determined by the method of McCord andFridovich. McCord et al., J. Biol. Chem., 244: 6049-6055 (1969).

Catalase was assayed by the method of Beers and Sizer. Beers et al., J.Biol. Chem., 195, pp. 133-140 (1952). The disappearance of peroxide isfollowed spectrophotometrically at 240 nm. The incubation mixture (3 ml)contains 50 μl of sample supernatant in 0.05 M potassium phosphate (pH7.0) and 0.02 M hydrogen peroxide. The decrease in absorbance recordedat 240 nm for 2 min. The rate of decrease in absorbance per min. iscalculated from the initial (45 sec.) linear portion of the curve.

The value of 0.0394 cm 2/μmol is used as the extinction coefficient ofH₂O₂

One unit of catalase is defined as the amount of enzyme which decomposes1 μmol of H₂O₂/min at 250° C. at pH 7.0 under the specified conditions.

Glutathione peroxidase was assayed as described by Carrillo et al.(Carrillo et al., Life Sci., 48:517-521 (1991)).

II. Results A. Assessment of Protandim I Toxicity

To assess any possible toxic effect of Protandim I supplementation,animals were weighed at the beginning and at the end of the study, and apercentage weight gain or loss was calculated for each. The most generalindication of toxicity is “failure to thrive,” which may manifest as adecreased rate of growth, or as an actual weight loss. FIG. 1 shows thatsupplementation actually caused modest but non-significant increases ingrowth rate. The absence of toxicity was evidenced as failure to thriveat any level of Protandim I supplementation, from the anticipated humandosage (1×) to a level of ten times that amount.

B. Protandim I Effect on SOD Activity

SOD activity was measured in the mice RBCs (FIG. 2), livers (FIG. 3) andbrains (FIG. 4) to assess the effect of Protandim I on this enzyme.Animals supplemented with Protandim I showed a dose-dependent increasein RBC SOD activity, as seen in FIG. 2. A significant 25% increase wasseen at the highest level of supplementation. It should be noted thatmature, circulating RBC do not contain nuclei, and therefore are notcapable of inducing new synthesis of enzymes once they enter thecirculation. RBC have a circulating lifespan of 120 days. Thus, duringthe 23 day course of the experiment about 20% of the RBC would have beenreplaced by maturing reticulocytes from the bone marrow. As the olderRBC are diluted out by the newly produced cells, one may predict thatthe increase in RBC SOD will proceed linearly with time until all cellshave been replaced at 120 days. Therefore, the predicted final RBC SODactivities expected (after 120 days) would be about 16%, at 1×, 44%, at3×, and 125% at 10× diet.

As detailed in FIG. 3, a dose-dependent increase in liver SOD activitywas observed. As shown in the figure, a significant increase was seen atall levels of supplementation: 13% at 1×, 23% at 3×, and 45% at 10×diet. Also, a significant increase in brain SOD activity was seen, butonly at the 3× level of Protandim I (FIG. 4). The percentage increasewas about 20%.

C. Protandim I Effect on CAT Activity

Animals supplemented with Protandim I showed little change in CAT. InRBC, there appeared to be a small dose-dependent increase, with anincrease in the 10× group that approached statistical significance(p=0.057) (FIG. 5). Based on the turnover of RBC as discussed above, onecan predict that the increase in RBC CAT would proceed linearly withtime until all cells have been replaced at 120 days. Therefore, thepredicted final RBC CAT activities expected after 120 days mightapproach 10% at 1×, 20% at 3×, and 40% at 10× diet.

Liver CAT was unchanged on 1× and 3× diets, but showed a smallnon-significant decrease in activity on the 10× diet (FIG. 6). Brainnormally contains very little catalase, and the small amounts detecteddid not change at any diet level.

D. Protandim I Effect on GPX Activity

Animals supplemented with Protandim I showed an unexpected decrease inGPX activity. In RBC, there appeared to be a small dose-dependentdecrease, with a 13% decrease in the 10× group that was notstatistically significant (FIG. 7). Based on the turnover of RBC asdiscussed above, one can predict that the decrease in RBC GPX wouldproceed linearly with time until all cells have been replaced at 120days. Therefore, the predicted decrease in RBC GPX activity expectedafter 120 days might approach 65% at 10× diet. There was a similardecrease in liver GPX that appeared to be dose-dependent, with a 40%decrease in the 10× group that was statistically significant at p<0.004(FIG. 8). Brain followed a similar pattern, with significant decreasesof 19% at 3× (p<0.03) and 23% at 10× (p=0.01 (FIG. 9).

E. Protandim I Effect on Lipid Peroxidation (TBARS)

One objective of Protandim treatment is to decrease oxidative stress.Our endpoint to assess oxidative stress in this study was TBARS. Animalssupplemented with Protandim I showed dramatic and highly significantdecreases in TBARS in all tissues studied. In plasma, there was adose-dependent decrease: 135% decrease in animals on the 1× diet; 62# onthe 3× diet; and 75% on the 10× diet. The changes in the 3× and 10×groups had high statistical significance: p=0.004 and p=0.0004,respectively (FIG. 10).

As shown in FIG. 11, in liver homogenates, there was a similardose-dependent decrease: a 34% decrease in animals on the 1× diet; 56%on the 3× diet; and 66% on the 10× diet. The changes in this tissue hadhigh statistical significance at all there diet concentrations (FIG.11). In brain homogenates, the effect of Protandim I was even morestriking: an 83% decrease was seen on the 1× diet (p<0.004); 94% on the3× diet (p<0.0001; and 97% on the 10× diet (p<=0.0001) (FIG. 12).

III. Summary of Protandim I's Effects

The present study established that herbal compositions of the presentinvention, e.g., Protandim I (as defined in Table 3), are a safe andeffective way of decreasing oxidative stress. The dosage defined as “1×”(16 mg/kg body weight) seems perfectly positioned for safety andefficacy. The major unexpected finding of the study was that, of thethree antioxidant enzymes measured, only SOD was clearly induced. CATwas unaffected, and GPX levels actually showed a significant decline.This study was based on claims in the literature that seven of the eightherbal ingredients upregulated SOD (Bhattacharya et al., Phytother,Res., 14:174-179 (2000); Soto et al., Comp Biochem. Physiol C. Toxicol.Pharmacol., 136:205-212 (2003); Bhattacharya et al., Indian J. Exp.Biol., 35:236-239 (1997); Bridi et al., Phytother. Res., 15:449-451(2001); Luczaj et al., J. Toxicol. Environ. Health A, 67:595-606 (2004);Reddy et al., Food Chem., Toxicol., 32:279-283 (1994); Singh et al.,Phytomedicine, 7:209-219 (2000); Naidu et al., Indian J. Exp. Biol.,40:894-900 (2002)), and that all eight upregulated CAT, and GPX(Bhattacharya et al., Phytother, Res., 14:174-179 (2000); Soto et al.,Comp Biochem. Physiol C. Toxicol. Pharmacol., 136:205-212 (2003);Bhattacharya et al., Indian J. Exp. Biol., 35:236-239 (1997); Bridi etal., Phytother. Res., 15:449-451 (2001); Luczaj et al., J. Toxicol.Environ. Health A, 67:595-606 (2004); Reddy et al., Food Chem.,Toxicol., 32:279-283 (1994); Singh et al., Phytomedicine, 7:209-219(2000); Naidu et al., Indian J. Exp. Biol., 40:894-900 (2002); Skukla etal., Phytother. Res., 13:50-54 (1999)).

FIG. 13 summarizes the Protandim-induced changes in antioxidant enzymesin blood, liver, and brain where clear and distinctly different patternsemerge. SOD was induced in a dose-dependent fashion, with the onlydeparture from the pattern being seen in brain with the 10× diet. As maybe seen in FIG. 14, brain TBARS was, in fact, nearly totally eliminatedon the 10× diet, suggesting that protection from oxidative stress wasmaximal.

FIG. 14 summarizes the effect of each Protandim I diet level on lipidperoxidation in each of the three tissues examined. There was a strongdose-dependency apparent, with no suggestion of any paradoxical increasein lipid peroxidation due to the well-established bell-shaped curve seenwith the effect of increasing SOD concentration on rate of lipidperoxidation. This is an important safety consideration. While it isclear that increasing SOD will be beneficial to individuals experiencinghigh oxidative stress, there was a possibility that increased SOD wouldbecome problematic to healthy individuals with little oxidative stress.This study, therefore, was designed to examine the ability of ProtandimI to decrease oxidative stress in young, normal mice, without causing aparadoxical increase in oxidative stress at the upper end of a ten-folddosage range. Other compositions of the invention likely share the samepositive health benefits when administered to a subject.

Example 2 Herbal Compositions of the Invention Alter Antioxidant Enzymesand Decreased Lipid Peroxidation in Human Subjects I. General Purposeand Design of Study

The purpose of this study was to observe the effects of herbalcompositions of the present invention on human subjects. An exemplarydietary supplement containing five herbal extracts, called Protandim IIwas administered to sixteen healthy human subjects ranging in age from20 to 78 years old. The composition of Protandim II is shown below inTable 5.

TABLE 5 Composition of Protandim II. Percent Weight Total ActiveIngredient Amount Active Ingredients B. monniera extract, 45% Bacosides150 mg 22.2 Milk Thistle extract, 70-80% 225 mg 33.3 SilymarinAshwagandha powder 150 mg 22.2 Turmeric extract, 95% Curcumin 75 mg 11.1Green tea, 98% polyphenols, 45% 75 mg 11.1 EGCG Total 675 mg 99.9

Subjects were assigned to two groups as defined in Table 6.

TABLE 6 Protandim II Dosing of Human Subjects No. of Age Dailysupplement of subjects range Protandim II Group 1 12 20 to 78 675 mgGroup 2 4 29 to 66 338 mg TOTAL 16

Group 1 had twelve subjects who received the full daily Protandim IIsupplement of 675 mg in a single daily capsule for 120 days. At 0, 30,and 120 days, blood was taken by venipuncture for analysis. Group 2 hadfour additional participants who received one-half as much Protandim(i.e., half dose), or 338 mg in a single daily capsule for 30 days.

II. Methods

The end points measured in tissue from human subjects includedactivities of the major antioxidant enzymes SOD and CAT in RBCs. Theextent of lipid peroxidation was assessed by measuring thiobarbituricacid reactive substances (TBARS) in plasma. In addition, uric acid wasmeasured in plasma because it is believed to be an endogenousantioxidant of some importance, especially with regard to scavenging andneutralizing the oxidant peroxynitrite. If oxidative stress weredecreased, one might expect a sparing effect on plasma uric acid levels.High sensitivity CRP was monitored as an indicator of inflammatoryactivity, and lipid profiles (total cholesterol, LDL, HDL, andtriglycerides) were assessed.

Thiobarbituric acid reactive substances, or TBARS, were determined bythe method of Ohkawa et al. (Ohkawa et al., Anal. Biochem., 95: 351-358(1979). The reaction mixture (total volume of 1 ml) contained 50 μl of8.1% sodium dodecyl sulfate, 0.375 ml of 20% acetic acid and 0.375 ml0.8% thiobarbituric acid, and 200 μl of plasma or tissue homogenatesupernate. The mixture was heated in boiling water for 1 hr, cooled withtap water and extracted with n-butanol/pyridine (15:1 v/v) by vortexingfor 1-2 min. The mixture was then centrifuged at 500-1000 g for 10 minor until a good aqueous-organic phase separation occurred. The organicphase was removed and its absorbance at 532 nm was measured against areaction mixture blank. A standard curve was prepared with1,1,3,3-tetramethoxypropane, and TBARS are reported as molarequivalents.

Superoxide dismutase activity was determined by the method of McCord andFridovich. McCord et al., J. Biol. Chem., 244: 6049-6055 (1969).

Catalase was assayed by the method of Beers and Sizer. Beers et al., J.Biol. Chem., 195, pp. 133-140 (1952). The disappearance of peroxide isfollowed spectrophotometrically at 240 nm. The incubation mixture (3 ml)contains 50 μl of sample supernatant in 0.05 M potassium phosphate (pH7.0) and 0.02 M hydrogen peroxide. The decrease in absorbance recordedat 240 nm for 2 min. The rate of decrease in absorbance per min. iscalculated from the initial (45 sec.) linear portion of the curve.

The value of 0.0394 cm2/μmol is used as the extinction coefficient ofH2O2

One unit of catalase is defined as the amount of enzyme which decomposes1 μmol of H2O2/min at 250° C. at pH 7.0 under the specified conditions.

High sensitivity C-reactive protein, uric acid, and lipid profileanalyses (total cholesterol, LDL, HDL, triglycerides) were performed bythe clinical chemistry laboratory of the University Hospital/Universityof Colorado Health Sciences Center, Denver, Colo.

Glutathione peroxidase was assayed as described by Carrillo et al.(Carrillo et al., Life Sci., 48:517-521 (1991)).

III. Results A. Assessment of Protandim II Toxicity or Side Effects

All subjects were instructed to report any suspected adverse reaction orside effect (such as nausea, vomiting, headache, gastrointestinaldiscomfort, diarrhea, constipation, itching, etc.) to the investigatorsimmediately, and to discontinue use of the supplement. No such reactionsor side effects were reported. No toxicity or evidence of other unwantedpharmacological effects of Protandim II were noted at either level ofsupplementation.

B. Effect of Protandim II on Lipid Peroxidation and TBARS

One objective of Protandim II treatment was to decrease oxidativestress. Our endpoint to assess oxidative stress in this study was TBARS,which measures a family of lipid peroxidation products (mostly lipidhydroperoxides) which break down during the analysis to yieldmalondialdehyde, which reacts with thiobarbituric acid to yield thechromophore measured at 532 nm. The TBA assay has been somewhatcontroversial, criticized by some for lack of specificity because ityields higher values than gas chromatographic methods specific formalondialdehyde. The ability of the TBA test to collectively measurelipid peroxidation products, including precursors that will continue tobreak down to yield malondialdehyde, is a strength of the TBA assay.Gutteridge et al., J. Appl. Biochem., 5:293-299 (1983); Liu et al.,Anal. Biochem. 245:161-166 (1997). It is clear that native fatty acidsin the absence of lipid peroxides do not undergo significantperoxidation during the acid-heating stage of the TBA test. Gutteridgeet al., J. Appl. Biochem., 5:293-299 (1983). Furthermore, the TBA testis the most widely used in the literature to assess lipid peroxidation,enabling easy comparison with the largest number of studies from otherlaboratories. In particular, a recent study found plasma TBARS to be apredictor of cardiovascular events in patients with established heartdisease, independently of traditional risk factors and inflammatorymarkers. Walter et al., J. Am. Coll. Cardiol., 44:1996-2002 (2004).

FIG. 15 (panel A and panel B) illustrates the age-related increase inplasma TBARS in sixteen healthy human subjects ranging in age from 20 to78 years old, prior to supplementation with Protandim II (solidcircles). As shown in FIG. 15, panel A, while there is substantialscatter around the linear regression line, there is a strong correlationwith age (R²=0.602) with the oldest individuals showing valuesapproximately three-fold higher than the youngest individuals. Aftersupplementation with Protandim II for 30 days (675 mg/day, n=11), thevalues for plasma TBARS declined as shown by the gray squares. Thescatter is remarkably less, and the correlation with age virtuallydisappears (R²=0.082). After 30 days, the average TBARS concentrationwas 0.95±0.04 μM. Nine of these individuals were assayed after 120 daysof supplementation (open circles), showing no further change (0.99±0.05μM; R²=0.012). All subjects showed decreased TBARS after 30 days onProtandim II. The age-related increase in lipid peroxidation productsdisappeared with Protandim II supplementation. The changes weremaintained at 120 days, with results indistinguishable from those at 30days.

C. Effect of Protandim II on SOD Activity

Subjects supplemented with Protandim II showed a statisticallysignificant increase of 22% (n=9, p=0.04) in erythrocyte SOD activityafter 120 days of supplementation, as seen in FIG. 16. Erythrocyte SODat day 0 was 378±17 U/ml and 460±18 U/ml by day 120. It should be notedthat mature, circulating erythrocytes do not contain nuclei, andtherefore are not capable of inducing new synthesis of enzymes once theyenter the circulation. Erythrocytes have a circulating lifespan of 120days. Thus, during the 120 day course of the experiment 100% of the redcells would have been replaced by maturing reticulocytes from the bonemarrow.

D. Effect of Protandim II on CAT Activity

Subjects supplemented with Protandim II showed a statisticallysignificant increase of 57% (n=9, p=0.001) in erythrocyte CAT activityafter 120 days of supplementation, as seen in FIG. 17. Erythrocyte CATat day 0 was 104,000±8,000 U/ml and 163,000±8,000 U/ml by day 120. Thesame considerations regarding turnover and replacement of erythrocytesapply to catalase as discussed above for SOD.

E. Effect of Protandim II on Select Blood Parameters

Subjects supplemented with Protandim II showed an increase of 4.6% inplasma uric acid concentration after 30 days of supplementation, butthis increase did not achieve statistical significance. Because uricacid serves as an endogenous antioxidant, it was anticipated that uricacid levels might rise as a result of increased SOD activity, whichwould lead to lower levels of peroxynitrite production. Uric acid isthought to scavenge the oxidant peroxynitrite.

Three subjects entered the study with elevated CRP levels, there was atrend towards reduction of these levels with Protandim IIsupplementation. As shown in FIG. 18, the CRP dropped an average of 24%after 30 days of Protandim II supplementation. The CRP levels continuedto decline at 60-70 days, illustrating a decrease of greater thanaverage of 30%-33% decline in CRP levels over the time tested. Nosignificant changes were seen in total cholesterol, LDL, HDL, ortriglycerides.

F. Effect of Low-Dose Protandim II (338 mg/day)

To assess whether the suggested human supplement of 675 mg/day might bemore than needed to achieve the desired reduction in oxidative stress,four subjects were given a lower dose of 338 mg/day for 30 days. Bloodwas drawn from these individuals on day 0, 5, 12 and 30 to provideadditional information regarding the time required for the reduction inoxidative stress to manifest. FIG. 19 shows that the response of plasmaTBARS is fairly rapid, with most of the change occurring by 5 to 12days. FIG. 20 shows that the lower dose of Protandim II was not quite aseffective as the full dose, lowering TBARS to an average value of1.29±0.14 μM (n=4) versus 0.95±0.04 μM (n=11). Using a one-tailedt-test, this difference was significant at p<0.03. This providesreassurance of the appropriateness of the full recommended dose ofProtandim of 675 mg/day is not an excessive dose.

IV. Discussion

The present study established that herbal compositions of the presentinvention, e.g., Protandim II (as defined in Table 5), are a safe andeffective way of decreasing oxidative stress in healthy human subjectsranging in age from 20 to 78. The dosage defined (675 mg/kg body weight)seems well-positioned for safety and efficacy. The age-dependentincrease in oxidative stress as measured by lipid peroxidation wasabolished. There was no evidence that the subjects showing the lowestinitial levels of oxidative stress were in any way compromised by themodest elevations of SOD and catalase that were achieved—an outcomeconsidered remote but theoretically possible due to our recognition thatthere is a bell-shaped dose-response curve to SOD. That is, problems canresult from too much SOD as well as from too little. The results fromthis study suggest that all subjects benefited from the ProtandimII-induced elevations of SOD and catalase activities.

The effects of Protandim II may go beyond direct induction of the SODand/or catalase genes. The antioxidant enzymes form a system of mutualprotection: superoxide inactivates both CAT and GPX, while hydrogenperoxide inactivates the cytosolic SOD. McCord, Free Radical Biol. Med.,4:9-14 (1988); Kono et al., J. Biol. Chem., 257:5751-5754 (1982); Blumet al., Arch. Biochem. Biophys., 240:500-508 (1985); Bray et al.,Biochem. J., 139:43-48 (1974). Thus, in a system experiencingsubstantial oxidative stress the entire group of antioxidant enzymes maybe subject to partial inactivation by the unscavenged concentrations ofsuperoxide and hydrogen peroxide. If, under these conditions, SOD alonewere induced, the concentration of superoxide would decrease, allowingpartial recovery of the activities of CAT and GPX as they escape fromsuperoxide-mediated inactivation. Thus, it would appear that all threeenzymes were induced. This might be expected only when starting underconditions of substantial oxidative stress. Under normal conditions,there may be little inactivation of CAT and GPX taking place, so theinduction of SOD might have less effect on the activities of the othertwo enzymes. Another factor to consider is that 4-hydroxynonenal, aproduct of lipid peroxidation, serves to induce the synthesis of GPX.Larini et al., Free Radic. Res., 38:509-516 (2004). If the induction ofSOD results in a lowering of the rate of lipid peroxidation, then theconcentration of 4-hydroxynonenal would fall and one might expect thatless GPX would be synthesized. This, in fact, is what we observed: SODwas induced, lipid peroxidation was inhibited, and GPX activity fell.Other compositions of the invention likely share the same positivehealth benefits when administered to a subject.

Example 3 Herbal Compositions of the Invention Normalize Blood Pressurein Human Subjects

Hypertension has been recognized as a multi-factorial trait resultingfrom the effect of a combination of environmental and genetic factors,including excess dietary salt or alcohol intake, stress, age, geneticsand family history, obesity, physical inactivity, as well as highsaturated fat diet. During the past few years, however, a large amountof information has been collected on the vascular inflammation,indicating that inflammation may involve in the initiation as well asdevelopment of hypertension. Evidence from animal models as well aspatients, have indicated that hypertension, an established major riskfactor for coronary artery disease, has been suggested to exertpro-inflammatory actions through the increased expression of severalmediators, including leukocyte adhesion molecules, chemokines, specificgrowth factors, heat shock proteins, endothelin-1, and angiotensin.Endothelial dysfunction as well as increased serum levels of C-reactiveprotein are observed in patients with hypertension (see, Li., J., MedHypotheses. Is hypertension an inflammatory disease? 2005;64(2):236-240). Assessment of changes in gene expression associated withincreased arterial stiffness and gene polymorphisms that increase therisk for vascular stiffening suggests that components of therenin-angiotensin system, matrix metalloproteinases, intracellularsignaling, and extracellular matrix components may all be involved inthis process. Interventions aimed at these targets may reduce vascularstiffness, lower systolic blood pressure, decrease the prevalence ofISH, and improve outcomes for patients (particularly older patients)with hypertension or other CV conditions (see, Schiffrin E L., Vascularstiffening and arterial compliance: implications for systolic bloodpressure. Am J Hypertens. 2004 December; 17(12 Pt 2):39S-48S). Thecompositions of the present invention thus provide novel therapeuticstrategies to decrease the morbidity as well as mortality ofhypertension, and alleviated hypertensive target organ damage.

Subject #1 was a 50 year old man whose blood pressure readings prior toadministration of a composition of the invention ranged from 140 to 150systolic over 90 to 100 diastolic. Following daily ingestion of 1500 mgof a composition containing B. monniera extract, Milk Thistle extract70-80%, Ashwagandha powder, Turmeric extract 95%, Gotu kola powder, Aloevera powder, Green tea (98% Polyphenols 45% EGCG), and Ginko biloba leafextract for 14 days, the subjects blood pressure was measured as 126systolic over 74 diastolic. The approximate daily dosage of each of thecomponents of the herb-containing composition was as follows: B.monniera extract (200 mg), Milk Thistle extract 70-80% (300 mg),Ashwagandha powder (200 mg), Turmeric extract (95%) (100 mg), Gotu kolapowder (200 mg), Aloe vera powder (200 mg), Green tea (98% Polyphenols45% EGCG) (100 mg), and Ginko biloba leaf extract (200 mg).

Subject # 2 was a 44 year old woman whose blood pressure readings priorto any treatment were approximately 165 systolic over 113 diastolic to160 systolic over 103 diastolic. The subject began taking ATENOLOL 25mg/day (9 months). The ATENOLOL medication reduced the subject's bloodpressure to 142 systolic over 98 diastolic to 135 systolic over 96diastolic, however, the subject's diastolic measurement never got downbelow 91 (the lowest on ATENOLOL). Following daily ingestion of 1,500 mgof an herb-containing composition containing B. monniera extract, MilkThistle extract 70-80%, Ashwagandha powder, Turmeric extract 95%, Gotukola powder, Aloe vera powder, Green tea (98% Polyphenols 45% EGCG), andGinko biloba leaf extract for 21 days (while ATENOLOL treatment wascontinued), the subjects blood pressure was measured as 136 systolicover 70 diastolic. Thus, a drop in diastolic pressure of more than 20 mmwas observed during this period (see FIG. 21, panel A). Thereafter,blood pressure remained constant as both treatments continued. Theapproximate daily dosage of each of the components of theherb-containing composition was as follows: B. monniera extract (200mg), Milk Thistle extract 70-80% (300 mg), Ashwagandha powder (200 mg),Turmeric extract (95%) (100 mg), Gotu kola powder (200 mg), Aloe verapowder (200 mg), Green tea (98% Polyphenols 45% EGCG) (100 mg), andGinko biloba leaf extract (200 mg).

Subject #3 was a 49 year old man whose blood pressure readings prior toadministration of a composition of the invention was 135 systolic over78 diastolic. Following daily ingestion of 1500 mg of a compositioncontaining B. monniera extract, Milk Thistle extract 70-80%, Ashwagandhapowder, Turmeric extract 95%, Gotu kola powder, Aloe vera powder, Greentea (98% Polyphenols 45% EGCG), and Ginko biloba leaf extract for 7days, the subjects blood pressure was measured as 117 systolic over 75diastolic. The approximate daily dosage of each of the components of theherb-containing composition was as follows: B. monniera extract (200mg), Milk Thistle extract 70-80% (300 mg), Ashwagandha powder (200 mg),Turmeric extract (95%) (100 mg), Gotu kola powder (200 mg), Aloe verapowder (200 mg), Green tea (98% Polyphenols 45% EGCG) (100 mg), andGinko biloba leaf extract (200 mg).

Subject # 4 was a 59 year old man whose blood pressure readings prior toany treatment were approximately 128 systolic over 75 diastolic.Following daily ingestion of 1000 mg of a composition containing B.monniera extract, Milk Thistle extract 70-80%, Ashwagandha powder,Turmeric extract 95%, Gotu kola powder, Aloe vera powder, Green tea (98%Polyphenols 45% EGCG), and Ginko biloba leaf extract for 21 days, thesubject's blood pressure was measured as 125 systolic over 61 diastolic.Thus, a drop in diastolic pressure of about 14 mm was observed duringthis period (see FIG. 21, panel B). After 21 days, blood pressureremained constant as treatment continued. The approximate daily dosageof each of the components of the herb-containing composition was asfollows: B. monniera extract (134 mg), Milk Thistle extract 70-80% (200mg), Ashwagandha powder (134 mg), Turmeric extract (95%) (67 mg), Gotukola powder (134 mg), Aloe vera powder (134 mg), Green tea (98%Polyphenols, 45% EGCG) (67 mg), and Ginko biloba leaf extract (134 mg).

Example 4 Compositions of the Invention Prevents or Alleviates MigraineHeadaches in Human Subjects

Migraines afflict about 24 million people in the United States. They mayoccur at any age, but usually begin between the ages of 10 and 40 anddiminish after age 50. Some people experience several migraines a month,while others have only a few migraines throughout their lifetime.Approximately 75% of migraine sufferers are women.

A migraine is a throbbing or pulsating headache that is often one sided(unilateral) and associated with nausea; vomiting; sensitivity to light,sound, and smells; sleep disruption; and depression. Attacks are oftenrecurrent and tend to become less severe as the migraine sufferer ages.Migraines are classified according to the symptoms they produce. The twomost common types are migraine with aura and migraine without aura. Lesscommon types include the following: Basilar artery migraine;Carotidynia; Headache-free migraine; Opthalmoplegic migraine; Statusmigraine

The cause of migraine is unknown. The condition may result from a seriesof reactions in the central nervous system caused by changes in the bodyor in the environment. There is often a family history of the disorder,suggesting that migraine sufferers may inherit sensitivity to triggersthat produce inflammation in the blood vessels and nerves around thebrain, causing pain.

A trigger is any stimulus that initiates a process or reaction. Commonlyidentified migraine triggers include the following: Alcohol (e.g., redwine); environmental factors (e.g., weather, altitude, time zonechanges); foods that contain caffeine (e.g., coffee, chocolate);monosodium glutamate (MSG; found in Chinese food); and nitrates (e.g.,processed foods, hot dogs); glare; hormonal changes in women; hunger;lack of sleep, medications (over-the-counter and prescription); perfume;stress.

In the present study, a female subject (woman) who routinely suffersmigraine headaches was administered a composition of the invention toassess the affect of the composition on the incidence of her migraineheadaches. The subject ingested a composition containing B. monnieraextract, Milk Thistle extract 70-80%, Ashwagandha powder, Turmericextract 95%, Gotu kola powder, Aloe vera powder, Green tea (98%Polyphenols 45% EGCG), and Ginko biloba leaf extract for 30 days. Theapproximate daily dosage of each of the components of theherb-containing composition was as follows: B. monniera extract (134mg-200 mg), Milk Thistle extract 70-80% (200 mg-300 mg), Ashwagandhapowder (134 mg-200 mg), Turmeric extract (95%) (67 mg-100 mg), Gotu kolapowder (134 mg-200 mg), Aloe vera powder (134 mg-200 mg), Green tea (98%Polyphenols 45% EGCG) (67 mg-100 mg), and Ginko biloba leaf extract (134mg-200 mg). Following the initiation of administration of thecomposition of the invention, the subject has not had a migraineheadache. The composition of the invention is therefore useful toprevent or alleviate migraine headaches in a subject.

Similarly, the compositions of the present invention are useful toprevent or treat headaches in a subject associated with high altitude(e.g., acute mountain sickness (AMS)). In the context of a recentascent, a headache, with any one or more of the following symptoms above2500 meters (8000 feet) qualifies a subject for the diagnosis of AMS:loss of appetite, nausea, or vomiting; fatigue or weakness; dizziness orlight-headedness; difficulty sleeping; confusion; staggering gait. Thecompositions of the present invention are also useful to prevent ortreat headaches, both acute and chronic.

Example 5 Embodiments of the Herb-Containing Composition of theInvention

In one embodiment of the method preventing, alleviating or treatingoxidative stress in a subject, an herb-containing composition containingB. monniera extract, Milk Thistle extract, 70-80% Silymarin, Ashwagandhapowder, Green tea, 98% polyphenols, 45% EGCG, and Turmeric extract, and95% Curcumin is administered at least daily to a subject. In oneembodiment, the herb-containing composition of the invention contains B.monniera extract, 45% bacosides, Milk Thistle extract, 70-80% Silymarin,Ashwagandha powder, Green tea, 98% polyphenols, 45% EGCG, and turmericextract, 95% curcumin in concentrations as detailed below in Table 7.

TABLE 7 Composition of Protandim II Weight Percent (wt %) Total ActiveIngredient Quantity Active Ingredients Bacopa monniera, extract 150 mg22.2 45% bacosides Milk Thistle extract, 225 mg 33.3 70-80% silymarinAshwagandha powder 150 mg 22.2 Green tea, 98% 75 mg 11.1 polyphenols,45% EGCG Turmeric extract, 95% 75 mg 11.1 curcumin Total 675 mg 99.9

In one embodiment, the herb-containing composition of the inventioncontains from about 5 wt % to about 50 wt % B. monniera extract (45%bacosides) of the total weight of active ingredients. In one embodiment,the herb-containing composition of the invention contains from about 10wt % to about 30 wt % B. monniera extract (45% bacosides) of the totalweight of active ingredients. In one embodiment, the herb-containingcomposition of the invention contains at least about 22 wt % B. monnieraextract (45% bacosides) of the total weight of active ingredients.

In one embodiment, the herb-containing composition of the inventioncontains from about 5 wt % to about 60 wt % milk thistle extract(70%-80% silymarin) of the total weight of active ingredients. In oneembodiment, the herb-containing composition of the invention containsfrom about 10 wt % to about 50 wt % milk thistle extract (70%-80%silymarin) of the total weight of active ingredients. In one embodiment,the herb-containing composition of the invention contains at least about33 wt % milk thistle extract (70%-80% silymarin) of the total weight ofactive ingredients.

In one embodiment, the herb-containing composition of the inventioncontains from about 5 wt % to about 50 wt % Ashwagandha powder of thetotal weight of active ingredients. In one embodiment, theherb-containing composition of the invention contains from about 10 wt %to about 30 wt % Ashwagandha extract of the total weight of activeingredients. In one embodiment, the herb-containing composition of theinvention contains at least about 22 wt % Ashwagandha extract of thetotal weight of active ingredients.

In one embodiment, the herb-containing composition of the inventioncontains from about 2.5 wt % to about 25 wt % turmeric extract (95%curcumin) of the total weight of active ingredients. In one embodiment,the herb-containing composition of the invention contains from about 5wt % to about 15 wt % turmeric extract (95% curcumin) of the totalweight of active ingredients. In one embodiment, the herb-containingcomposition of the invention contains at least about 11 wt % turmericextract (95% curcumin) of the total weight of active ingredients.

In one embodiment, the herb-containing composition of the inventioncontains from about 2.5 wt % to about 25 wt % green tea (98%polyphenols, 45% EGCG) of the total weight of active ingredients. In oneembodiment, the herb-containing composition of the invention containsfrom about 5 wt % to about 15 wt % green tea (98% polyphenols, 45% EGCG)of the total weight of active ingredients. In one embodiment, theherb-containing composition of the invention contains at least about 11wt % green tea (45% polyphenols) of the total weight of activeingredients.

In some embodiments of the herb-containing composition of the presentinvention, the composition comprises the active ingredients assummarized above in Table 7 as well as Gotu kola powder, Ginko bilobaleaf extract and Aloe vera powder as detailed below in Table 8.

TABLE 8 Composition of Protandim I. Percent Weight Total ActiveIngredient Amount Active Ingredients Bacopa monniera extract, 45% 150 mg12.77 bacosides Milk Thistle extract, 70-80% 275 mg 23.4 silymarinAshwagandha powder 150 mg 12.77 Gotu kola power 150 mg 12.77 Turmericextract, 95% curcumin 75 mg 6.38 Green tea 98% polyphenols, 45% 75 mg6.38 EGCG Ginko biloba leaf extract 150 mg 12.77 Aloe vera powder 150 mg12.77 Total 1175 mg 100

In one embodiment, the herb-containing composition of the inventioncontains from about 5 wt % to about 50 wt % B. monniera extract (45%bacosides) of the total weight of active ingredients. In one embodiment,the herb-containing composition of the invention contains from about 10wt % to about 30 wt % B. monniera extract (45% bacosides) of the totalweight of active ingredients. In one embodiment, the herb-containingcomposition of the invention contains at least about 22 wt % B. monnieraextract (45% bacosides) of the total weight of active ingredients. Inone embodiment, the herb-containing composition of the inventioncontains at least about 12 wt % B. monniera extract (45% bacosides) ofthe total weight of active ingredients.

In one embodiment, the herb-containing composition of the inventioncontains from about 5 wt % to about 60 wt % milk thistle extract(70%-80% silymarin) of the total weight of active ingredients. In oneembodiment, the herb-containing composition of the invention containsfrom about 10 wt % to about 50 wt % milk thistle extract (70%-80%silymarin) of the total weight of active ingredients. In one embodiment,the herb-containing composition of the invention contains at least about33 wt % milk thistle extract (70%-80% silymarin) of the total weight ofactive ingredients. In one embodiment, the herb-containing compositionof the invention contains at least about 23 wt % milk thistle extract(70%-80% silymarin) of the total weight of active ingredients.

In one embodiment, the herb-containing composition of the inventioncontains from about 5 wt % to about 50 wt % Ashwagandha powder of thetotal weight of active ingredients. In one embodiment, theherb-containing composition of the invention contains from about 10 wt %to about 30 wt % Ashwagandha extract of the total weight of activeingredients. In one embodiment, the herb-containing composition of theinvention contains at least about 22 wt % Ashwagandha extract of thetotal weight of active ingredients. In one embodiment, theherb-containing composition of the invention contains at least about 12wt % Ashwagandha extract of the total weight of active ingredients.

In one embodiment, the herb-containing composition of the inventioncontains from about 2.5 wt % to about 25 wt % turmeric extract (95%curcumin) of the total weight of active ingredients. In one embodiment,the herb-containing composition of the invention contains from about 5wt % to about 15 wt % turmeric extract (95% curcumin) of the totalweight of active ingredients. In one embodiment, the herb-containingcomposition of the invention contains at least about 11 wt % turmericextract (95% curcumin) of the total weight of active ingredients. In oneembodiment, the herb-containing composition of the invention contains atleast about 6 wt % turmeric extract (95% curcumin) of the total weightof active ingredients.

In one embodiment, the herb-containing composition of the inventioncontains from about 2.5 wt % to about 25 wt % green tea (98%polyphenols, 45% EGCG) of the total weight of active ingredients. In oneembodiment, the herb-containing composition of the invention containsfrom about 5 wt % to about 15 wt % green tea (98% polyphenols, 45% EGCG)of the total weight of active ingredients. In one embodiment, theherb-containing composition of the invention contains at least about 11wt % green tea (45% polyphenols) of the total weight of activeingredients. In one embodiment, the herb-containing composition of theinvention contains at least about 6 wt % green tea (45% polyphenols) ofthe total weight of active ingredients.

In one embodiment the composition, the Gotu kola powder is present at aconcentration from at least about 5 weight percent to about 50 weightpercent of the total dry weight of active ingredients of thecomposition. In one embodiment of the composition, the Gotu kola powderis present at a concentration from at least about 10 weight percent toabout 30 weight percent of the total dry weight of active ingredients ofthe composition. In one embodiment of the composition, the Gotu kolapowder extract is present at a concentration at least about 12 weightpercent of the total dry weight of active ingredients of thecomposition.

In one embodiment of the composition, the Ginko biloba leaf extract ispresent at a concentration from at least about 5 weight percent to about50 weight percent of the total dry weight of active ingredients of thecomposition. In one embodiment of the composition, the Ginko biloba leafextract is present at a concentration from at least about 10 weightpercent to about 30 weight percent of the total dry weight of activeingredients of the composition. In one embodiment of the composition,the Ginko biloba leaf extract is present at a concentration at leastabout 12 weight percent of the total dry weight of active ingredients ofthe composition.

In one embodiment of the composition, the Aloe vera powder is present ata concentration from at least about 5 weight percent to about 50 weightpercent of the total dry weight of active ingredients of thecomposition. In one embodiment of the composition, the Aloe vera powderis present at a concentration from at least about 10 weight percent toabout 30 weight percent of the total dry weight of active ingredients ofthe composition. In one embodiment of the composition, the Aloe verapowder is present at a concentration of at least about 12 weight percentof the total dry weight of active ingredients of the composition.

In some embodiments, the herb-containing compositions of the inventionare formulated to contain at least one excipient such as, e.g., calciumcarbonate; croscarmellose sodium; dicalcium phosphate; magnesiumstearate; microcrystalline cellulose; modified cellulose; silica; andstearic acid.

EQUIVALENTS

While the invention has been described in connection with the specificembodiments thereof, it will be understood that it is capable of furthermodification. Furthermore, this application is intended to cover anyvariations, uses, or adaptations of the invention, including suchdepartures from the present disclosure as come within known or customarypractice in the art to which the invention pertains, and as fall withinthe scope of the appended claims.

1. A composition comprising: (a) at least about 150 milligrams Bacopamonniera extract, wherein said Bacopa monniera extract having 20 percentbacosides or greater; at least about 225 milligrams Silybum marianum(milk thistle) extract, wherein said milk thistle extract is havingbetween about 70 percent and about 80 percent silymarin; at least about150 mg Withania somnifera (ashwagandha) powder; at least about 75milligrams Camellia sinensis (green tea) extract; wherein said green teaextract is comprised of 98 percent polyphenols; said polyphenolscomprised of 45 percent (−)-epigallocatechin gallate; at least about 75milligrams Curcuma longa (turmeric) extract; wherein said turmericextract is comprised of 95 percent curcumin; wherein the compositionincreases the enzyme activity level of at least one antioxidant enzymeselected from the group consisting of: superoxide dismutase and catalaseand decreases the plasma concentration level of thiobarbituric acidreactive chemical species, when administered in an effective amount to amammalian subject in need thereof.
 2. The composition according to claim1, wherein the composition is formulated as an oral dosage form.
 3. Thecomposition according to claim 2, wherein the oral dosage form isselected from the group consisting of: a tablet; capsule; and caplet. 4.The composition according to claim 1, wherein the composition includesone or more excipients selected from the group consisting of calciumcarbonate; croscarmellose sodium; dicalcium phosphate; magnesiumstearate; microcrystalline cellulose; modified cellulose; silica; andstearic acid.